US20040172681A1

US20040172681A1 - Elevation of oil levels in brassica plants

Info

Publication number: US20040172681A1
Application number: US10/742,350
Authority: US
Inventors: Toni Voelker; Dale Val; Thomas Savage
Original assignee: Individual
Current assignee: Monsanto Technology LLC
Priority date: 2002-12-19
Filing date: 2003-12-19
Publication date: 2004-09-02
Also published as: WO2004056848A2; AU2003303242A1; CA2509227C; US7405344B2; EP1583417B1; AU2003303242B2; CA2509227A1; EP1583417A2; ATE527875T1; WO2004056848A3; EP1583417A4

Abstract

This present invention provides a method for increasing oil levels in the tissues of Brassica sp. plants by expression of a heterologous multifunctional fatty acid synthase (mfFAS) within the plant. In one embodiment, the present invention provides isolated nucleic acid molecules encoding mfFAS enzymes from various sources for this purpose, and vectors and plants containing same.

Description

This patent application claims the benefit of U.S. Provisional Patent Application No. 60/435,197, filed Dec. 19, 2002, incorporated herein by reference.[0001]

The present invention relates to the fields of nucleic acid chemistry and agricultural biotechnology. In particular, the present invention is directed at the identification of nucleic acids that encode proteins useful for increasing oil levels in Brassica plants and creating Brassica plants that include such nucleic acids.

Brassica plants are a source of polyunsaturated oils. While tissues of most Brassica plant species contain little oil, the cultivation of certain plant types, over many acres, permit large quantities of Brassica plant oils to be produced. If the oil content of these Brassica plants could be increased, then Brassica plant oils could be produced more efficiently.

Higher plants such as Brassica synthesize fatty acids via a common metabolic pathway involving the co-factor ACP and the fatty acid synthase (FAS) enzyme complex. The FAS complex consists of about 8 separate enzymes that catalyze 30 or more individual reaction steps, all of which, in plants, are located in the plastids. In developing seeds, for example, where fatty acids are stored, the fatty acid synthase (FAS) enzyme complex is located in the plastids, synthesizes the fatty acids therein, and then the fatty acids are transported to the cytosol in accordance with energy needs there.

Certain workers have attempted to increase or modulate the oil content of plants. For example, U.S. Pat. No. 6,268,550 to Gengenbach et al., provides maize acetyl CoA carboxylase nucleic acids for altering the oil content of plants. Additionally, U.S. Pat. No. 5,925,805 to Ohlrogge et al., provides an Arabidopsis acetyl CoA carboxylase gene that can be used to increase the oil content of plants.

SUMMARY OF THE INVENTION

A need exists for a method to increase the oil content of Brassica plants and seeds. Moreover, it would be more energy efficient to provide the plant with a capability to synthesize fatty acids in the cytosol directly. This present invention provides a Brassica sp. plant comprising a heterologous nucleic acid encoding a multifunctional fatty acid synthase. In one embodiment, the plant further comprises a second nucleic acid encoding a phosphopantetheine:protein transferase. In one preferred embodiment the Brassica plant produces increased oil levels in the seed tissue as a result of the multifunctional fatty acid synthase. In another preferred embodiment the multifunctional fatty acid synthase is substantially located in the cytosol of the plant cell.

This present invention further provides a method for increasing oil levels in the tissues of a Brassica sp. plant by expressing a gene encoding a multifunctional fatty acid synthase on either a single or multiple polypeptide chains. In one preferred embodiment of this present invention, the gene encodes a cytosol-targeted mfFAS. Preferably, the source of the mfFAS is selected from the group consisting of bacteria, fungae, planta, mycoplasma, and the like; more preferably, the source of the mfFAS is bacteria or fungae; most preferably, the source of the mfFAS is bacteria. In another embodiment of this present invention, the expression of the mfFAS gene is in the seed tissue of the Brassica plant, preferably resulting in the accumulation of oil in the seed.

In another embodiment the present invention provides plant transformation vectors for Brassica containing a mfFAS gene, as well as transformed Brassica plants and seeds are also provided.

In another embodiment, the present invention provides a method of producing a Brassica oil, comprising the steps of: a) growing an oilseed Brassica plant, the genome of which contains a nucleic acid molecule encoding a multifunctional fatty acid synthase, to produce oil-containing seeds; and b) extracting oil from the seeds. In another embodiment, the present invention provides a method of producing a Brassica oil, comprising the steps of: a) growing an oilseed Brassica plant, the genome of which contains a nucleic acid molecule encoding a phosphopantetheine:protein transferase, to produce oil-containing seeds; and b) extracting oil from the seeds.

DESCRIPTION OF THE FIGURES

FIG. 1 provides a map of the plasmid pMON70058 that contains the 8 KB fasA gene from [0010] Brevibacterium ammoniagenes.
FIG. 2 provides an alignment of the fasA [0011] Brevibacterium ammoniagenes nucleic acid sequence [SEQ ID NO: 1] provided herein with a published fasA Brevibacterium ammoniagenes nucleic acid sequence (Stuible et al., J. Bacteriol., 178:4787, 1996). A number of differences at the DNA level were observed.
FIG. 3 provides an alignment of the fasA [0012] Brevibacterium ammoniagenes amino acid sequence [SEQ ID NO: 1] provided herein with a published fasA Brevibacterium ammoniagenes amino acid sequence (Stuible et al., J. Bacteriol., 178:4787, 1996). A number of differences at the protein level were observed.
FIG. 4 illustrates FasA enzyme activity of the cloned fasA gene from [0013] B. ammoniagenes. The FasA enzyme activity was determined as outlined in Kawaguchi et al., Methods in Enzymology, 71:120-127 (1981) for partially purified enzyme preparations from B. ammoniagenes (B.a.), for an untransformed E. coli strain VCS257 (E.c.), and for the same strain transformed with the ppt1 expressing plasmid (E.c.+P), the fasA cosmid (E.c.+FA), or the ppt1 expressing plasmid and the fasA cosmid (E.c.+P+FA).
FIG. 5 provides a schematic representation of the preparation of pMON75201 as well as a map of pMON75201. [0014]
FIG. 6 shows the results of the analyses of R2 seed from events generated from the transformation of canola explants with the vector pMON75201. [0015]
FIG. 7 shows the statistical analysis of the oil results from positive and negative isolines of event BN_G1216.[0016]

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is a DNA encoding FasA from [0017] Brevibacterium ammoniagenes.
SEQ ID NO: 2 is a protein known as FasA from [0018] Brevibacterium ammoniagenes.
SEQ ID NO: 3 is a DNA encoding a phosphopantetheine:protein transferase (PPT1) enzyme from [0019] B. ammoniagenes.
SEQ ID NO: 4 is a protein known as phosphopantetheine:protein transferase (PPT1) enzyme from [0020] B. ammoniagenes.
SEQ ID NO: 5 is a nucleic acid used as a PCR primer. [0021]
SEQ ID NO: 6 is a nucleic acid used as a PCR primer. [0022]
SEQ ID NO: 7 is a nucleic acid used as a PCR primer. [0023]
SEQ ID NO: 8 is a nucleic acid used as a PCR primer. [0024]
SEQ ID NO: 9 is a nucleic acid used as a PCR primer. [0025]
SEQ ID NO: 10 is a nucleic acid used as a PCR primer. [0026]
SEQ ID NO: 11 is a nucleic acid used as a PCR primer. [0027]
SEQ ID NO: 12 is a nucleic acid used as a PCR primer. [0028]
SEQ ID NO: 13 is a nucleic acid used as a PCR primer. [0029]
SEQ ID NO: 14 is a nucleic acid used as a PCR primer. [0030]
SEQ ID NO: 15 is a protein known as [0031] fatty acid synthase 1 of Schizosaccharomyces pombe; NCBI Accession No. CAB54157.
SEQ ID NO: 16 is a DNA encoding fatty acid synthase subunit beta of [0032] Schizosaccharomyces pombe.
SEQ ID NO: 17 is a protein known as fatty acid synthase subunit alpha of [0033] Schizosaccharomyces pombe; NCBI Accession No. D83412.
SEQ ID NO: 18 is a protein known as fatty acid synthase subunit beta of [0034] Saccharomyces cerevisiae; NCBI Accession No. CAA82025.
SEQ ID NO: 19 is a protein known as fatty acid synthase subunit alpha of [0035] Saccharomyces cerevisiae; NCBI Accession No. CAA97948.
SEQ ID NO: 20 is a protein known as fatty acid synthase subunit beta of [0036] Candida albicans; NCBI Accession No. CAA52907.
SEQ ID NO: 21 is a DNA encoding fatty acid synthase subunit alpha of [0037] Candida albicans; NCBI Accession No. L29063.
SEQ ID NO: 22 is a protein known as fatty acid synthase subunit alpha of [0038] Candida albicans; NCBI Accession No. L29063.
SEQ ID NO: 23 is a protein known as fatty acid synthase of [0039] Mycobacterium tuberculosis H37Rv; NCBI Accession No. CAB06201.
SEQ ID NO: 24 is a protein known as fatty acid synthase of [0040] Mycobacterium leprae; NCBI Accession No. CAB39571.
SEQ ID NO: 25 is a protein known as fatty acid synthase of [0041] Caenorhabditis elegans; NCBI Accession No. NP492417.
SEQ ID NO: 26 is a DNA encoding fatty acid synthase (FAS) of [0042] Rattus norvegicus; NCBI Accession No. X13415.
SEQ ID NO: 27 is a protein known as fatty acid synthase (FAS) of [0043] Rattus norvegicus; NCBI Accession No. X13415.
SEQ ID NO: 28 is a DNA encoding fatty acid synthase (FAS) of chicken ([0044] Gallus gallus); NCBI Accession No. J03860 M22987.
SEQ ID NO: 29 is a protein known as fatty acid synthase (FAS) of chicken ([0045] Gallus gallus); NCBI Accession No. J03860 M22987.
SEQ ID NO: 30 is a DNA encoding fatty acid synthase (FAS) of [0046] Mycobacterium bovis; NCBI Accession No. U36763.
SEQ ID NO: 31 is a protein known as fatty acid synthase (FAS) of [0047] Mycobacterium bovis; NCBI Accession No. U36763.
SEQ ID NO: 32 is a DNA encoding a phosphopantetheine:protein transferase (sfp gene product) enzyme from [0048] Bacillus subtilis; NCBI Accession No. X63158.
SEQ ID NO: 33 is a protein known as phosphopantetheine:protein transferase (sfp gene product) enzyme from [0049] Bacillus subtilis; NCBI Accession No. X63158.
SEQ ID NO: 34 is a DNA encoding a phosphopantetheine:protein transferase (gsp gene product) enzyme from [0050] Brevibacillus brevis (ATCC 9999); NCBI Accession No. X76434.
SEQ ID NO: 35 is a protein known as phosphopantetheine:protein transferase (gsp gene product) enzyme from [0051] Brevibacillus brevis (ATCC 9999); NCBI Accession No. X76434.
SEQ ID NO: 36 is a DNA encoding a phosphopantetheine:protein transferase (entD gene product) enzyme from [0052] Escherichia coli; NCBI Accession No. D90700.
SEQ ID NO: 37 is a protein known as phosphopantetheine:protein transferase (entD gene product) enzyme from [0053] Escherichia coli; NCBI Accession No. D90700.
SEQ ID NO: 38 is a DNA encoding a phosphopantetheine:protein transferase (pptA gene product) enzyme from [0054] Streptomyces verticillus; NCBI Accession No. AF210311.
SEQ ID NO: 39 is a protein known as phosphopantetheine:protein transferase (pptA gene product) enzyme from [0055] Streptomyces verticillus; NCBI Accession No. AF210311.
SEQ ID NO: 40 is a DNA encoding an α-aminoadipate reductase small subunit (lys5 gene product) enzyme from [0056] Saccharomyces cerevisiae; NCBI Accession No. U32586.
SEQ ID NO: 41 is a protein known as the small subunit (lysS gene product) of an α-aminoadipate reductase from [0057] Saccharomyces cerevisiae; NCBI Accession No. U32586.
SEQ ID NO: 42 is a DNA encoding an open reading frame o195 from [0058] Escherichia coli; NCBI Accession No. U00039.
SEQ ID NO: 43 is a protein encoded by open reading frame o195 from [0059] Escherichia coli; NCBI Accession No. U00039.

Definitions

The following definitions are provided as an aid to understanding the detailed description of the present invention. [0060]
The phrases “coding sequence,” “coding region,” “structural sequence,” and “structural nucleic acid sequence” refer to a physical structure comprising an orderly arrangement of nucleotides. The nucleotides are arranged in a series of triplets that each form a codon. Each codon encodes a specific amino acid. Thus, the coding sequence, coding region, structural sequence, and structural nucleic acid sequence encode a series of amino acids forming a protein, polypeptide, or peptide sequence. The coding sequence, coding region, structural sequence, and structural nucleic acid sequence may be contained within a larger nucleic acid molecule, vector, or the like. In addition, the orderly arrangement of nucleotides in these sequences may be depicted in the form of a sequence listing, figure, table, electronic medium, or the like. [0061]
The phrases “DNA sequence,” “nucleic acid sequence,” and “nucleic acid molecule” refer to a physical structure comprising an orderly arrangement of nucleotides. The DNA sequence or nucleotide sequence may be contained within a larger nucleotide molecule, vector, or the like. In addition, the orderly arrangement of nucleic acids in these sequences may be depicted in the form of a sequence listing, figure, table, electronic medium, or the like. [0062]
The term “expression” refers to the transcription of a gene to produce the corresponding mRNA and translation of this mRNA to produce the corresponding gene product (i.e., a peptide, polypeptide, or protein). [0063]
The phrase “expression of antisense RNA” refers to the transcription of a DNA to produce a first RNA molecule capable of hybridizing to a second RNA molecule, which second RNA molecule encodes a gene product that is desirably down-regulated. [0064]
The term “homology” refers to the level of similarity between 2 or more nucleic acid or amino acid sequences in terms of percent of positional identity (i.e., sequence similarity or identity). Homology also refers to the concept of similar functional properties among different nucleic acids or proteins. [0065]
The term “heterologous” refers to the relationship between 2 or more nucleic acid or protein sequences that are derived from different sources. For example, a promoter is heterologous with respect to a coding sequence if such a combination is not normally found in nature. In addition, a particular nucleic acid molecule may be “heterologous” with respect to a cell or organism into which it is inserted (i.e., does not naturally occur in that particular cell or organism). [0066]
The term “hybridization” refers to the ability of a first strand of nucleic acid to join with a second strand via hydrogen bond base pairing when the 2 nucleic acid strands have sufficient sequence complementarity. Hybridization occurs when the 2 nucleic acid molecules anneal to one another under appropriate conditions. [0067]
The phrase “operably linked” refers to the functional spatial arrangement of 2 or more nucleic acid regions or nucleic acid sequences. For example, a promoter region may be positioned relative to a nucleic acid sequence such that transcription of the nucleic acid sequence is directed by the promoter region. Thus, a promoter region is operably linked to the nucleic acid sequence. [0068]
In the context of the present invention, the terms “plant” or “plants” refer to plants of the species Brassica. [0069]
The terms “promoter” or “promoter region” refers to a nucleic acid sequence, usually found upstream (5′) to a coding sequence, that is capable of directing transcription of a nucleic acid sequence into mRNA. The promoter or promoter region typically provides a recognition site for RNA polymerase and the other factors necessary for proper initiation of transcription. As contemplated herein, a promoter or promoter region includes variations of promoters derived by inserting or deleting regulatory regions, subjecting the promoter to random or site-directed mutagenesis, and the like. The activity or strength of a promoter may be measured in terms of the amounts of RNA it produces, or the amount of protein accumulation in a cell or tissue, relative to a second promoter that is similarly measured. [0070]
The term “5′-UTR” refers to the untranslated region of DNA upstream, or 5′, of the coding region of a gene. [0071]
The term “3′-UTR” refers to the untranslated region of DNA downstream, or 3′, of the coding region of a gene. [0072]
The phrase “recombinant vector” refers to any agent by or in which a nucleic acid of interest is amplified, expressed, or stored, such as a plasmid, cosmid, virus, autonomously replicating sequence, phage, or linear single-stranded, circular single-stranded, linear double-stranded, or circular double-stranded DNA or RNA nucleotide sequence. The recombinant vector may be derived from any source and is capable of genomic integration or autonomous replication. [0073]
The phrase “regulatory sequence” refers to a nucleotide sequence located upstream (5′), within, or downstream (3′) with respect to a coding sequence. Transcription and expression of the coding sequence is typically impacted by the presence or absence of the regulatory sequence. [0074]
The phrase “substantially homologous” refers to 2 sequences that are at least about 90% identical in sequence, as measured by the CLUSTAL W method in the Omiga program, using default parameters (Version 2.0; Accelrys, San Diego, Calif.). [0075]
The term “transformation” refers to the introduction of nucleic acid into a recipient host. The term “host” refers to bacteria cells, fungi, animals or animal cells, plants or seeds, or any plant parts or tissues including plant cells, protoplasts, calli, roots, tubers, seeds, stems, leaves, seedlings, embryos, and pollen. [0076]
As used herein, the phrase “transgenic Brassica plant” refers to a Brassica plant having an introduced nucleic acid stably introduced into a genome of that plant, for example, the nuclear or plastid genomes. [0077]
As used herein, the phrase “substantially purified” refers to a molecule separated from substantially all other molecules normally associated with it in its native state. More preferably, a substantially purified molecule is the predominant species present in a preparation. A substantially purified molecule may be greater than about 60% free, preferably about 75% free, more preferably about 90% free, and most preferably about 95% free from the other molecules (exclusive of solvent) present in the natural mixture. The phrase “substantially purified” is not intended to encompass molecules present in their native state. [0078]

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a multifunctional fatty acid synthase (“mfFAS”) that encodes the enzymatic functions required to synthesize palmitoyl (16:0) CoA, stearoyl (18:0) CoA, and oleoyl (18:1) CoA, the fatty acids used as precursors for other long chain saturated and unsaturated fatty acids. Obtaining nucleic acid sequences capable of producing increased oil content in Brassica plants is problematic because many non-associated, monofunctional enzymes are used to make fatty acids in Brassica plants. Accordingly, cloning and genetic manipulation of plant fatty acid synthases (“FASs”) would require isolation and coordinated expression of at least 8 separate genes. In particular, plant fatty acid synthesis depends on availability of the following plastid-localized FAS enzymes: Malonyl-CoA:ACP transacylase, β-ketoacyl-ACP synthase III, β-ketoacyl-ACP synthase I, β-ketoacyl-ACP synthase II, β-ketoacyl-ACP reductase, β-hydroxyacyl-ACP dehydratase, enoyl-ACP reductase, and stearoyl-ACP desaturase. For movement of the end-product, acyl-ACP, from the plastid to the cytosol of the cell, two more enzymatic activities are required: acyl-ACP thioesterase and acyl-CoA synthase. [0079]
However, the present invention solves this problem by providing a multifunctional fatty acid synthase that encodes all of the FAS enzymatic functions in a single, long polypeptide chain or in two chains that combine together, which may be employed in the cytosol or plastid, preferably in both, more preferably in the cytosol. Such a multifunctional fatty acid synthase is surprisingly effective in Brassica plants even though its structure is so dissimilar from plant endogenous fatty acid synthases. Most preferably, the mfFAS of the present invention is employed in the cytosol of a Brassica plant, and in this way the need for an acyl carrier protein (“ACP”) in fatty acid synthesis and the enzymes acyl-ACP thioesterase and acyl-CoA synthase is removed. Accordingly, not only does the present invention remove the need to clone at least 8 different genes to accomplish altered fatty acid synthesis in a Brassica plant, but when the mfFAS is employed in the cytosol, it replaces the function of 11 different plant gene products. [0080]
Fatty Acid Synthases: Fatty acid synthases are among the functionally most complex multienzyme systems known, which can be formed from a single polypeptide or multiple polypeptides. For mfFASs formed of a single polypeptide, there are multiple regions included thereon that perform the various enzymatic activities; such regions are referred to as “domains.” Multifunctional fatty acid synthases formed of multiple polypeptides include various FAS domains as well, which require the interaction of the constituent polypeptides to function. Such polypeptides, whether a multi-domain or single-domain polypeptide, can be isolated from an organism, or can be generated by combining domains or parts of domains together at the nucleic acid level using conventional recombinant technology. Accordingly, recombinant chimeric nucleic acids that combine some mfFAS domains from one source with the remainder of the mfFAS domains from one or more second sources are preferred embodiments of the present invention. In the same fashion, the nucleic acid sequences in a mfFAS gene that encodes a particular domain can be replaced with a homologous nucleic acid sequence from a second source that encodes the same domain. [0081]
Fatty acid synthases usually comprise a set of 8 different functional domains and catalyze more than about 30 individual reaction steps. Two structurally distinct classes of fatty acid synthases exist. Type I fatty acid synthases are multifunctional synthases, commonly found in non-plant eukaryotes and in a few bacterial species. Type II fatty acid synthases constitute a set of separate, monofunctional polypeptides that are found in most bacteria and in the plastids of higher plants. These polypeptides must properly assemble into a multimeric complex before the synthase becomes active. The fatty acid synthase from some bacteria, such as [0082] Brevibacterium ammoniagenes, is unlike plant and animal synthases in that it has a ninth catalytic activity (Seyama and Kawaguchi (1987), in Dolthin et al., (eds.), Pyridine Nucleotide Coenzymes: Chemical, Biochemical and Medical Aspects, vol. 2B, Wiley, NY, pp. 381-431), the 3-hydroxydecanoyl β,γ-dehydratase, which enables synthesis of both saturated and unsaturated fatty acids.
For transgenic purposes, type I multifunctional fatty acid synthases may have certain advantages over the type II “monofunctional” fatty acid synthases. For example, the type I multifunctional fatty acid synthases may have greater stability and/or better-coordinated expression. Addition of a single polypeptide specific for one of the enzymatic fatty acid synthase activities to a plant by transgenic means may not provide overproduction of the entire fatty acid synthase complex because there may not be sufficient endogenous amounts of the other non-transgenic FAS polypeptides to substantially increase levels of the functional complex. In contrast, nucleic acids encoding a type I multifunctional fatty acid synthase can reliably be used to overproduce all of the enzymatic functions of fatty acid synthase. [0083]
According to the present invention, nucleic acids encoding one or more of the separate domains from a type II monofunctional fatty acid synthase can be fused or linked to provide a synthetic multifunctional fatty acid synthase that can generate high oil levels when expressed within a host, such as, for example, a Brassica plant cell, plant tissue, or seed. Such a fused, synthetic multifunctional fatty acid synthase can be made by fusing or linking the separate enzymatic functions associated with the various polypeptides of type II fatty acid synthases by chemically linking the nucleic acids that encode the various polypeptides. The overall sequence of such a synthetic gene generally aligns with that of a type I multifunctional fatty acid synthase. Using such sequence alignments, the spacing and orientation of polypeptides that contain the various fatty acid synthase activities can be adjusted or modified by altering the lengths of linking DNA between coding regions to generate a synthetic multifunctional fatty acid synthase DNA construct that optimally aligns with a natural type I multifunctional fatty acid synthase gene. [0084]
The fatty acid synthase polypeptides of the present invention can therefore encode more than one of the enzymes associated with fatty acid synthase, such as, for example, 2 through and including 9, thereby enabling up to the same 9 catalytic activities as are found in the mfFAS of [0085] Brevibacterium ammoniagenes. Any of the enzymes involved in the various steps of fatty acid synthesis can be joined. The first step in initiation stage of fatty acid synthesis is the carboxylation of the 2-carbon acetyl-CoA to form the 3-carbon β-ketoacid malonyl-CoA by acetyl-CoA carboxylase (ACCase). The ACCase step is irreversible, so once this step is accomplished, the resultant carbon compound is committed to fatty acid synthesis. All subsequent steps are catalyzed by the FAS. Malonyl-ACP is synthesized from malonyl-CoA and ACP by the enzyme malonyl-CoA:ACP transacylase. An acetyl moiety from acetyl-CoA is joined to a malonyl-ACP in a condensation reaction catalyzed by β-ketoacyl-ACP synthase III. Elongation of acetyl-ACP to 16- and 18-carbon fatty acids involves the cyclical action of the following sequence of reactions. After acetyl-CoA is condensed with malonyl-ACP using β-ketoacyl-ACP synthase, a β-ketoacyl-ACP is formed. The keto group on the β-ketoacyl-ACP is then reduced to an alcohol by β-ketoacyl-ACP reductase. The alcohol is removed in a dehydration reaction to form an enoyl-ACP by β-hydroxyacyl-ACP dehydratase. Finally, the enoyl-ACP is reduced to form the elongated saturated acyl-ACP by enoyl-ACP reductase.
The enzyme β-ketoacyl-ACP synthase I catalyzes elongation up to palmitoyl-ACP (C16:0), which is generally the end product from which other types of fatty acids are made. The enzyme β-ketoacyl-ACP synthase II catalyzes the final elongation of palmitoyl-ACP to stearoyl-ACP (C18:0). [0086]
Common plant unsaturated fatty acids, such as oleic, linoleic, and α-linolenic acids, originate from the desaturation of stearoyl-ACP to form oleoyl-ACP (C18:1) in a reaction catalyzed by a soluble plastid enzyme, A-9 desaturase (also often referred to as “stearoyl-ACP desaturase”). Molecular oxygen is required for desaturation and reduced ferredoxin serves as an electron co-donor. [0087]
Hence, the present invention contemplates polypeptides encoding several functions, for example, those relating to acyl carrier protein, malonyl CoA-ACP acyltransferase, β-ketoayl-ACP synthase III, β-ketoayl-ACP reductase, β-hydroxyacyl-ACP dehydratase, enoyl-ACP reductase, β-ketoacyl-ACP synthase I, β-ketoacyl-ACP synthase II, and A-9 desaturase. [0088]
In one embodiment, the present invention provides an isolated mfFAS polypeptide from a species of the group consisting of Brevibacterium ammoniagenes, [0089] Schizosaccharomyces pombe, Saccharomyces cerevesiae, Candida albicans, Mycobacterium tuberculosis, Caenorhabditis elegans, Rattus norvegicus, Gallus gallus, Lipomyces starkeyi, Rhodosporidium toruloides, and Mycobacterium bovis. Preferably, the mfFAS polypeptide is isolated from Brevibacterium ammoniagenes, Schizosaccharomyces pombe, Saccharomyces cerevesiae, Candida albicans; more preferably, the mfFAS polypeptides is isolated from Brevibacterium ammoniagenes. Such mfFAS polypeptides include one selected from the group consisting of SEQ ID NOs: 2, 15, 17, 18, 19, 20, 22, 23, 24, 25, 27, 29, and 31. Preferably, the mfFAS polypeptide used in the context of the present invention is one selected from the group consisting of SEQ ID NOs: 2, 15, 17, 18, 19, 20, and 22; more preferably, the mfFAS is SEQ ID NO: 2. Any of the aforementioned mfFAS polypeptides functions to increase the oil content of Brassica plant tissues.
mfFAS Nucleic Acids: The present invention uses nucleic acids that encode multifunctional fatty acid synthases, which are used in the context of the present invention for increasing the oil content of Brassica plant tissues. Such nucleic acids can encode a type I multifunctional fatty acid synthase that has been isolated from an organism. Preferred organisms from which nucleic acids encoding mfFAS can be isolated include, without limitation: bacteria, preferably Brevibacteria and Bacilli; fungae, preferably Saccharomycetes, Schizosaccharomycetes, [0090] Lipomyces starkeyi, Rhodosporidium toruloides, or Candidae; mycobacteria; nematodes, preferably Caenorhabdites; and mammals, preferably rat or chicken. Alternatively, the nucleic acids can encode a multifunctional fatty acid synthase that has been recombinantly generated to contain a fusion of 2 or more regions that encode monofunctional enzymatic domains that facilitate 2 or more of the steps required to make a fatty acid.
In one embodiment, the present invention uses an isolated nucleic acid that encodes a protein having mfFAS activity, which nucleic acid is selected from the group consisting of SEQ ID NOs: 1, 16, 21, 26, 28, and 30, and complements thereof, and nucleic acids having at least about 70% sequence identity thereof. Preferred nucleic acid is SEQ ID NO: 1, 16, 21, 26, 28, or 30; more preferred is SEQ ID NO: 1. The percent sequence identity of included nucleic acids in the group is preferably at least about 75%, more preferably at least about 80%, yet more preferably at least about 85%, and yet more preferably at least about 90%; even more preferably at least about 95%; and most preferably at least about 98%. The nucleic acids of the present invention can be isolated from any species that has a multifunctional fatty acid synthase, including without limitation [0091] Brevibacterium ammoniagenes (source of SEQ ID NO: 1), Schizosaccharomyces pombe (source of SEQ ID NO: 16), Saccharomyces cerevesiae, Candida albicans (source of SEQ ID NO: 21), Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium bovis (source of SEQ ID NO: 30), Caenorhabditis elegans, rat (source of SEQ ID NO: 26), and chicken (source of SEQ ID NO: 28). In a preferred embodiment, the present invention provides a nucleic acid that encodes mfFAS from Brevibacterium ammoniagenes, Schizosaccharomyces pombe, Saccharomyces cerevesiae, or Candida albicans; more preferably, the mfFAS polypeptides are isolated from Brevibacterium ammoniagenes.
In yet another embodiment, the present invention uses a nucleic acid that encodes a multifunctional fatty acid synthase having an amino acid sequence comprising a protein selected from the group consisting of SEQ ID NOs: 2, 15, 17, 18, 19, 20, 22, 23, 24, 25, 27, 29, and 31. Preferably, the protein is SEQ ID NO: 2. The present invention also uses the set of nucleic acids that includes those nucleic acids that are at least about 80% identical to those that encode SEQ ID NO: 2, 15, 17, 18, 19, 20, 22, 23, 24, 25, 27, 29, or 31; more preferably, the set of nucleic acids are at least about 85% identical to one or more of the nucleic acids that encode one or more of the identified SEQ ID NOs; yet more preferably, at least about 90% identical; even more preferably, at least about 95% identical; and most preferably, at least about 98% identical. [0092]
The present invention also uses vectors containing such multifunctional fatty acid synthase nucleic acids. As set forth in further detail hereinbelow, preferred nucleic acids include appropriate regulatory elements operably linked thereto that facilitate efficient expression of the inventive nucleic acids in a host, including without limitation, Brassica plant hosts. Vectors useful in the context of the present invention can include such regulatory elements. [0093]
In a preferred embodiment of the present invention, the nucleic acid molecules of the present invention encode enzymes that are allelic to those defined. As used herein, a mutant enzyme is any enzyme that contains an amino acid that is different from the amino acid in the same position of an enzyme of the same type. [0094]
The nucleic acids and vectors described herein need not have the exact nucleic acid sequences described herein. Instead, the sequences of these nucleic acids and vectors can vary, so long as the nucleic acid either performs the function for which it is intended or has some other utility, for example, as a nucleic acid probe for complementary nucleic acids. For example, some sequence variability in any part of a multifunctional fatty acid synthase nucleic acid is permitted so long as the mutant or variant polypeptide or polypeptides retains at least about 10% of the fatty acid synthase (FasA) activity observed under similar conditions for an analogous wild type fatty acid synthase enzyme, more preferably, the polypeptide(s) retain at least about 25% of the FasA activity; more preferably at least about 50% of the FasA activity; even more preferably, at least about 75% of the FasA activity; and yet more preferably, at least about 90% of the FasA activity. Most preferably, the aforementioned sequence variability results in increased FasA activity. In a preferred embodiment, the comparison of enzymatic activity is with the wild type [0095] Brevibacterium ammoniagenes fatty acid synthase [SEQ ID NO: 2].
Fragment and variant nucleic acids, for example, of SEQ ID NO: 1, are also encompassed by the present invention. Nucleic acid “fragments” encompassed by the present invention are of 3 general types. First, fragment nucleic acids that are not full length but do perform their intended function (fatty acid synthesis) are encompassed within the present invention. Second, fragments of nucleic acids identified herein that are useful as hybridization probes, but generally are not functional for fatty acid synthesis, are also included in the present invention. And, third, fragments of nucleic acids identified herein can be used in suppression technologies known in the art, such as, for example, anti-sense technology or RNA inhibition (RNAi), which provides for reducing carbon flow in a plant into oil, making more carbon available for protein or starch accumulation, for example. Thus, fragments of a nucleotide sequence, such as SEQ ID NO: 1, 16, 21, 26, 28, or 30, without limitation, may range from at least about 15 nucleotides, at least about 17 nucleotides, at least about 18 nucleotides, at least about 20 nucleotides, at least about 50 nucleotides, at least about 100 nucleotides, or more. In general, a fragment nucleic acid of the present invention can have any upper size limit so long as it is related in sequence to the nucleic acids of the present invention but does not include the full length. [0096]
In another embodiment, the present invention provides DNA molecules comprising a sequence encoding a consensus amino acid sequence, and complements thereof. In another aspect, the present invention provides DNA molecules comprising a sequence encoding a polypeptide comprising a conserved fragment of an amino acid consensus sequence. The present invention includes the use of consensus sequence and fragments thereof in transgenic Brassica plants, other organisms, and for other uses including those described below. [0097]
As used herein, “variants” have substantially similar or substantially homologous sequences when compared to reference or wild type sequence. For nucleotide sequences that encode proteins, variants also include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the reference protein. Variant nucleic acids also include those that encode polypeptides that do not have amino acid sequences identical to that of the proteins identified herein, but which encode an active protein with conservative changes in the amino acid sequence. [0098]

As is known by one of skill in the art, the genetic code is “degenerate,” meaning that several trinucleotide codons can encode the same amino acid. This degeneracy is apparent from Table 1.

	TABLE 1


	2^nd Position

1^stPosition	T	C	A	G	3^rdPosition

T	TTT = Phe	TCT = Ser	TAT = Tyr	TGT = Cys	T
T	TTC = Phe	TCC = Ser	TAC = Tyr	TGC = Cys	C
T	TTA = Leu	TCA = Ser	TAA = Stop	TGA = Stop	A
T	TTG = Leu	TCG = Ser	TAG = Stop	TGG = Trp	G
C	CTT = Leu	CCT = Pro	CAT = His	CGT = Arg	T
C	CTC = Leu	CCC = Pro	CAC = His	CGC = Arg	C
C	CTA = Leu	CCA = Pro	CAA = Gln	CGA = Arg	A
C	CTG = Leu	CCG = Pro	CAG = Gln	CGG = Arg	G
A	ATT = Ile	ACT = Thr	AAT = Asn	AGT = Ser	T
A	ATC = Ile	ACC = Thr	AAC = Asn	AGC = Ser	C
A	ATA = Ile	ACA = Thr	AAA = Lys	AGA = Arg	A
A	ATG = Met	ACG = Thr	AAG = Lys	AGG = Arg	G
G	GTT = Val	GCT = Ala	GAT = Asp	GGT = Gly	T
G	GTC = Val	GCC = Ala	GAC = Asp	GGC = Gly	C
G	GTA = Val	GCA = Ala	GAA = Gln	GGA = Gly	A
G	GTG = Val	GCG = Ala	GAG = Gln	GGG = Gly	G

Hence, many changes in the nucleotide sequence of the variant may be silent and may not alter the amino acid sequence encoded by the nucleic acid. Where nucleic acid sequence alterations are silent, a variant nucleic acid will encode a polypeptide with the same amino acid sequence as the reference nucleic acid. Therefore, a particular nucleic acid of the present invention also encompasses variants with degenerate codon substitutions, and complementary sequences thereof, as well as the sequence explicitly specified by a SEQ ID NO as set forth herein. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the reference codon is replaced by any of the codons for the amino acid specified by the reference codon. In general, the third position of one or more selected codons can be substituted with mixed-base and/or deoxyinosine residues as disclosed by Batzer et al., [0100] Nucleic Acid Res., 19:5081 (1991) and/or Ohtsuka et al., J. Biol. Chem., 260:2605 (1985); Rossolini et al., Mol. Cell. Probes, 8:91 (1994).
A host cell often displays a preferred pattern of codon usage. Structural nucleic acid sequences are preferably constructed to utilize the codon usage pattern of the particular host cell. This generally enhances the expression of the structural nucleic acid sequence in a transformed host cell. Any disclosed nucleic acid or amino acid sequence may be modified to reflect the preferred codon usage of a host cell or organism in which they are contained. Modification of a structural nucleic acid sequence for optimal codon usage in plants is described in U.S. Pat. No. 5,689,052, which is incorporated herein by reference. In a preferred embodiment, the present invention includes nucleic acids that encode mfFAS and that are codon-optimized in a Brassica plant. In a preferred embodiment the plants are of the Brassica species, and most preferably [0101] Brassica napus (canola).
However, the present invention is not limited to silent changes in the present nucleotide sequences but also includes variant nucleic acid sequences that conservatively alter the amino acid sequence of a polypeptide of the present invention. Because it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence and, of course, its underlying DNA coding sequence and, nevertheless, a protein with like properties can still be obtained. It is thus contemplated by the inventors that various changes may be made in the peptide sequences of the proteins or fragments of the present invention, or corresponding DNA sequences that encode the peptides, without appreciable loss of their biological utility or activity. According to the present invention, then, variant and reference nucleic acids of the present invention may differ in the encoded amino acid sequence by one or more substitutions, additions, insertions, deletions, fusions, and truncations, which may be present in any combination, so long as an active mfFAS protein is encoded by the variant nucleic acid. Such variant nucleic acids will not encode exactly the same amino acid sequence as the reference nucleic acid, but have conservative sequence changes. It is known that codons capable of coding for such conservative amino acid substitutions are known in the art. [0102]
Another approach to identifying conservative amino acid substitutions require analysis of the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, [0103] J. Mol. Biol., 157:105-132, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant polypeptide, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, [0104] J. Mol. Biol., 157:105-132, 1982); these are isoleucine (+4.5), valine (+4.2), leucine (+3.8), phenylalanine (+2.8), cysteine/cystine (+2.5), methionine (+1.9), alanine (+1.8), glycine (−0.4), threonine (−0.7), serine (−0.8), tryptophan (−0.9), tyrosine (−1.3), proline (−1.6), histidine (−3.2), glutamate (−3.5), glutamine (−3.5), aspartate (−3.5), asparagine (−3.5), lysine (−3.9), and arginine (−4.5).
In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those that are within +1 are particularly preferred, and those within ±0.5 are even more particularly preferred. [0105]
It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. [0106]
As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0), lysine (+3.0), aspartate (+3.0±1), glutamate (+3.0+1), serine (+0.3), asparagine (+0.2), glutamine (+0.2), glycine (0), threonine (−0.4), proline (−0.5±1), alanine (−0.5), histidine (−0.5), cysteine (−1.0), methionine (−1.3), valine (−1.5), leucine (−1.8), isoleucine (−1.8), tyrosine (−2.3), phenylalanine (−2.5), and tryptophan (−3.4). [0107]
In making such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those that are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. [0108]
Variant nucleic acids with silent and conservative changes can be defined and characterized by the degree of homology to the reference nucleic acid. Preferred variant nucleic acids are “substantially homologous” to the reference nucleic acids of the present invention. As recognized by one of skill in the art, such substantially similar nucleic acids can hybridize under stringent conditions with the reference nucleic acids identified by SEQ ID NO herein. These types of substantially homologous nucleic acids are encompassed by this present invention. [0109]
Generally, nucleic acid derivatives and variants of the present invention will have at least about 90%, at least about 91%, at least about 92%, at least about 93%, or at least about 94% sequence identity to the reference nucleotide sequence defined herein. Preferably, nucleic acids of the present invention will have at least about 95%, at least about 96%, at least about 97%, or at least about 98% sequence identity to the reference nucleotide sequence defined herein. [0110]
Variant nucleic acids can be detected and isolated by standard hybridization procedures. Hybridization to detect or isolate such sequences is generally carried out under “moderately stringent” and preferably under “stringent” conditions. Moderately stringent hybridization conditions and associated moderately stringent and stringent hybridization wash conditions used in the context of nucleic acid hybridization experiments, such as Southern and Northern hybridization, are sequence dependent, and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular biology-Hybridization with Nucleic Acid Probes, [0111] page 1, Chapter 2, Overview of principles of hybridization and the strategy of nucleic acid probe assays, Elsevier, N.Y. (1993). See also, J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, pp. 9.31-9.58 (1989); J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY (3rd ed. 2001).
The present invention also provides methods for detection and isolation of derivative or variant nucleic acids encoding the proteins provided herein. The methods involve hybridizing at least a portion of a nucleic acid comprising any part of SEQ ID NO: 1, 16, 21, 26, 28, or 30, with respect to FAS-related sequences; and any part of SEQ ID NO: 3, 32, 34, 36, 38, 40, or 42, with respect to phosphopantetheine:protein transferase to a sample nucleic acid, thereby forming a hybridization complex; and detecting the hybridization complex. The presence of the complex correlates with the presence of a derivative or variant nucleic acid that can be further characterized by nucleic acid sequencing, expression of RNA and/or protein and testing to determine whether the derivative or variant retains activity. In general, the portion of a nucleic acid comprising any part of the aforementioned DNAs identified by SEQ ID NO used for hybridization is preferably at least about 15 nucleotides, and hybridization is under hybridization conditions that are sufficiently stringent to permit detection and isolation of substantially homologous nucleic acids; preferably, the hybridization conditions are “moderately stringent”; more preferably the hybridization conditions are “stringent”, as defined herein and in the context of conventional molecular biological techniques well known in the art. [0112]
Generally, highly stringent hybridization and wash conditions are selected to be about 5° C. lower than the thermal melting point (T[0113] _m) for the specific double-stranded sequence at a defined ionic strength and pH. For example, under “highly stringent conditions” or “highly stringent hybridization conditions” a nucleic acid will hybridize to its complement to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). By controlling the stringency of the hybridization and/or the washing conditions, nucleic acids having 100% complementary can be identified and isolated.
Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing) using, for example, moderately stringent conditions. Appropriate stringency conditions that promote DNA hybridization under moderately stringent conditions are, for example, about 2× sodium chloride/sodium citrate (SSC) at about 65° C., followed by a wash of 2×SSC at 20-25° C., are known to those skilled in the art and can be found in [0114] Current Protocols in Molecular Biology, John Wiley & Sons, NY, 6.3.1-6.3.6 (1989). Both temperature and salt may be varied, or either the temperature or the salt concentration may be held constant while the other variable is changed.
Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide, in which case hybridization temperatures can be decreased. Dextran sulfate and/or Denhardt's solution (50× Denhardt's is 5% Ficoll, 5% polyvinylpyrrolidone, 5% BSA) can also be included in the hybridization reactions. [0115]
Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 50% formamide, 5×SSC (20×SSC is 3M NaCl, 0.3 M trisodium citrate), 50 mM sodium phosphate, pH7, 5 mM EDTA, 0.1% SDS (sodium dodecyl sulfate), 5× Denhardt's with 100 μg/ml denatured salmon sperm DNA at 37° C., and a wash in 1× to 5×SSC (20×SSC=3.0 M NaCl and 0.3 M trisodium citrate), 0.1% SDS at 37° C. Exemplary moderate stringency conditions include hybridization in 40 to 50% formamide, 5×SSC 50 mM sodium phosphate, pH 7, 5 mM EDTA, 0.1% SDS, 5× Denhardt's with 100 μg/ml denatured salmon sperm DNA at 42° C., and a wash in 0.1× to 2×SSC, 0.1% SDS at 42 to 55° C. Exemplary high stringency conditions include hybridization in 50% formamide, 5×SSC, 50 mM sodium phosphate, pH 7.0, 5 mM EDTA, 0.1% SDS, 5× Denhardt's with 100 μg/ml denatured salmon sperm DNA at 42° C., and a wash in 0.1×SSC, 0.1% SDS at 60 to 65° C. [0116]
The degree of complementarity or homology of hybrids obtained during hybridization is typically a function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. The type and length of hybridizing nucleic acids also affects whether hybridization will occur and whether any hybrids formed will be stable under a given set of hybridization and wash conditions. For DNA-DNA hybrids, the T[0117] _mcan be approximated from the equation of Meinkoth and Wahl, Anal. Biochem., 138:267-284 (1984);
T_m=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L
where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The T[0118] _mis the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected for hybridization to derivative and variant nucleic acids having a T_mequal to the exact complement of a particular probe, less stringent conditions are selected for hybridization to derivative and variant nucleic acids having a T_mless than the exact complement of the probe.
In general, T[0119] _mis reduced by about 1° C. for each 1% of mismatching. Thus, T_m, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired sequence identity. For example, if sequences with greater than about 90% identity are sought, the T_mcan be decreased by about 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (T_m) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at about 1, about 2, about 3, or about 4° C. lower than the thermal melting point (T_m); moderately stringent conditions can utilize a hybridization and/or wash at about 6, about 7, about 8, about 9, or about 10° C. lower than the thermal melting point (T_m); low stringency conditions can utilize a hybridization and/or wash at about 11, about 12, about 13, about 14, about 15, or about 20° C. lower than the thermal melting point (T_m).
If the desired degree of mismatching results in a T[0120] _mof less than 45° C. (aqueous solution) or 32° C. (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, Part 1, Chapter 2, Elsevier, N.Y.; Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2, Greene Publishing and Wiley—Interscience, NY. See Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y. Using these references and the teachings herein on the relationship between T_m, mismatch, and hybridization and wash conditions, those of ordinary skill can generate variants of the present nucleic acids.
In another preferred embodiment of the present invention, the inventive nucleic acids are defined by the percent identity relationship between particular nucleic acids and other members of the class using analytic protocols well known in the art. Such analytic protocols include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif., or in the Omiga program version 2.0 Accelrys Inc., San Diego, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis.). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins et al., [0121] Gene, 73:237-244 (1988); Higgins et al., CABIOS, 5:151-153 (1989); Corpet et al., Nucleic Acids Res., 16:10881-90 (1988); Huang et al., CABIOS, 8:155-65 (1992); and Pearson et al., Meth. Mol. Biol., 24:307-331 (1994). The ALIGN program is based on the algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4:11-17 (1988). The BLAST programs of Altschul et al., J. Mol. Biol., 215:403 (1990), are based on the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. (U.S.A.), 87:2264-2268 (1990). To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al., Nucleic Acids Res., 25:3389 (1997). Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See, Altschul et al., supra. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, Henikoff & Henikoff, Proc. Natl. Acad. Sci. (U.S.A.), 89:10915, 1989). Alignment may also be performed manually by inspection.
For purposes of the present invention, comparison of nucleotide sequences for determination of percent sequence identity to the nucleic acid sequences disclosed herein is preferably made using the BLASTN program (version 1.4.7 or later) with its default parameters or any equivalent program. By “equivalent program” is intended any sequence comparison program that, for any 2 sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by the preferred program. [0122]
Isolation of Nucleic Acids Encoding Multifunctional Fatty Acid Synthases: Nucleic acids encoding a multifunctional fatty acid synthase can be identified and isolated by standard methods, as described by Sambrook et al., [0123] Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (1989). For example, a DNA sequence encoding a type I multifunctional fatty acid synthase can be identified by screening of a DNA or cDNA library generated from nucleic acid derived from a particular cell type, cell line, primary cells, or tissue. Examples of libraries useful for identifying and isolating a multifunctional fatty acid synthase include libraries made from the genomic DNA or cDNA of any organism encoding a type I fatty acid synthase, preferably a bacteria or non-plant eukaryote.
Screening for DNA fragments that encode a multifunctional fatty acid synthase can be accomplished by screening colonies or plaques from a genomic or cDNA library for hybridization to a probe of an available multifunctional fatty acid synthase from other organisms or by screening colonies or plaques from a cDNA expression library for binding to antibodies that specifically recognize a multifunctional fatty acid synthase. DNA fragments that hybridize to multifunctional fatty acid synthase probes from other organisms and/or colonies or plaques carrying DNA fragments that are immunoreactive with antibodies to multifunctional fatty acid synthase can be subcloned into a vector and sequenced and/or used as probes to identify other cDNA or genomic sequences encoding all or a portion of the desired multifunctional fatty acid synthase gene. Probes for isolation of multifunctional fatty acid synthase genes can also include DNA fragments of type II fatty acid synthase genes or antibodies to the type II proteins, as noted herein above. [0124]
A cDNA library can be prepared, for example, by random oligo priming or oligo dT priming. Plaques containing DNA fragments can be screened with probes or antibodies specific for multifunctional fatty acid synthase. DNA fragments encoding a portion of a multifunctional fatty acid synthase gene can be subcloned and sequenced and used as probes to identify a genomic multifunctional fatty acid synthase gene. DNA fragments encoding a portion of a multifunctional fatty acid synthase can be verified by determining sequence homology with other known multifunctional fatty acid synthase genes or by hybridization to multifunctional fatty acid synthase-specific messenger RNA. Once cDNA fragments encoding portions of the 5′, middle and 3′ ends of a multifunctional fatty acid synthase are obtained, they can be used as probes to identify and clone a complete genomic copy of the multifunctional fatty acid synthase gene from a genomic library. [0125]
Portions of the genomic copy or copies of an multifunctional fatty acid synthase gene can be sequenced and the 5′ end of the gene identified by standard methods, including either DNA sequence homology to other multifunctional fatty acid synthase genes or by RNAase protection analysis, as described by Sambrook et al., [0126] Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989). The 3′ and 5′ ends of the target gene can also be located by computer searches of genomic sequence databases using known fatty acid synthase coding regions. Once portions of the 5′ end of the gene are identified, complete copies of the multifunctional fatty acid synthase gene can be obtained by standard methods, including cloning or polymerase chain reaction (PCR) synthesis using oligonucleotide primers complementary to the DNA sequence at the 5′ end of the gene. The presence of an isolated full-length copy of the multifunctional fatty acid synthase gene can be verified by hybridization, partial sequence analysis, or by expression of the multifunctional fatty acid synthase enzyme.
Phosphopantetheine:Protein Transferases: During the process of fatty acid synthesis, the growing acyl chain is preferably covalently linked by a thioester bond to the cysteamine thiol of a phosphopantetheinyl (P-Pan) moiety, which is preferably attached at the other end to a specific serine residue of acyl carrier protein (ACP), in the case of type II FAS systems, or the ACP-domain of a type I FAS. This P-Pan moiety acts as a “swinging arm,” carrying the growing acyl chain between the active sites of the different enzymes or domains of the FAS complex. Accordingly, the transgenic mfFAS used in the context of the present invention is preferably phosphopantetheinylated, which phosphopantetheinylation is accomplished by a co-transformed gene that encodes a suitable PPTase or by a host PPTase that has sufficient substrate range of activity for the purpose of modifying the transgenic mfFAS. [0127]
The enzymatic post-translational attachment of the P-Pan group to an ACP protein or domain is carried out by a phosphopantetheinyl transferase (PPTase). Any suitable PPTase can be used in the context of the present invention, the suitability of which is determined by the ability of the PPTase to phosphopantetheinylate a mfFAS used herein. For example, the [0128] Brevibacterium ammoniagenes FasA protein [SEQ ID NO: 2] can be suitably combined with the PPTase from the same species, which is identified herein as SEQ ID NO: 4. In another embodiment of the present invention, the gene encoding the mfFAS includes its own PPTase activity, such as the mfFAS derived from yeast (e.g., SEQ ID NOs: 15-19), and thus the transgenic mfFAS is suitably modified to be active upon expression in the host. Particularly preferred PPTases have broad specificity, such as, for example, those referred to as being of the sfp-type, as further discussed hereinbelow. More preferred, the mfFAS employed in the context of the present invention is pantethenylated by an enzyme having PPTase activity that is native to the host Brassica plant into which the mfFAS transgene has been inserted.
A PPTase from [0129] Bacillus subtillis, the sfp gene product, has a remarkably broad range of substrate specificity, being able to phosphopantetheinylate non-native substrates both in vitro (Lambalot et al., Chem. Biol., 3:923-936, 1996) and in vivo (Mootz et al., J. Biol. Chem., 276:37389-37298, 2001); see FIG. 23 for recital of the sequences of the sfp gene and its product. Mootz and co-workers have shown that the sfp gene product not only complements heterologous PPTases, such as E. coli ACPS, but it in vivo phosphopantetheinylates all the different acceptor domains in natural host cells (e.g., Bacillus subtillis) that include ACP and PCP (petide carrier protein) of type I polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS) involved in secondary metabolism as well as the type II ACP protein required for fatty acid synthesis (primary metabolism). Indeed, this broad range of specificity appears to be a general feature of many sfp-type PPTases. Streptomyces verticullus svp PPTase (see FIG. 26), another sfp-type enzyme, was also found to be able to phosphopantetheinylate a broad range of substrates, including type I and II ACP and PCP domains from various Streptomyces species (Sanchez et al., Chem. Biol., 8:725-728, 2001). Other useful sfp-type PPTases include those found in Brevibacillus brevis (SEQ ID NO: 35), and Escherichia coli (SEQ ID NO: 36), which are listed here without any intention to limit the sfp-type PPTases that are usefully employed in the context of the present invention. Preferably, the Bacillus subtilis PPTase, that is the gene that encodes it, is used.
In the case of the multifunctional FasA and FasB proteins from [0130] Brevibacterium ammoniagenes, Stuible and co-workers found that the E. coli ACPS was unable to phosphopantetheinylate these type I FAS proteins either in vivo when the genes were introduced into E. coli or in vitro when mixed with the proteins. The B. ammoniagenes PPT1 protein was required to phosphopantetheinylate both of these type I FAS proteins (Stuible et al., Eur. J. Biochem., 248:481-487, 1997).
A preferred embodiment of the present invention relates to the use of an mfFAS that can be phosphopantetheinylated by a PPTase that is innate to a Brassica plant. An alternative preferred embodiment relates to the use of a PPTase specific for the introduced multifunctional FAS that is inserted in a Brassica plant, such as ppt1 in the case of the [0131] B. ammoniagenes fasA and fasB genes, which specific PPTase could be co-expressed in order to engineer functional multifunctional FAS expression in Brassica plants. As a further embodiment of the present invention, a PPTase of broad specificity, such as a sfp-type PPTase, may be co-expressed with a type II FAS gene in order to engineer functional multifunctional FAS expression in Brassica plants.
Expression Vectors and Cassettes: The expression vectors and cassettes of the present invention include nucleic acids encoding multifunctional fatty acid synthases. When inclusion of a heterologous phosphopantetheine protein transferase enzyme (PPTase) is desired, such expression vectors and cassettes can also include a nucleic acid encoding a PPTase that can post-translationally activate the multifunctional fatty acid synthase polypeptide. Alternatively, a separate expression vector or cassette can encode a phosphopantetheine protein transferase enzyme. One such PPTase is encoded by the [0132] B. ammoniagenes ppt1 gene. Other sources of PPTase having broad spectrum activity include: Bacillus subtilis, Brevibacillus brevis, Escherichia coli, Streptomyces verticullus, and Saccharomyces cerevisiae.
A transgene comprising a multifunctional fatty acid synthase can be subcloned into an expression vector or cassette, and fatty acid synthase expression can be detected and/or quantified. This method of screening is useful to identify transgenes providing for an expression of a multifunctional fatty acid synthase, and expression of a multifunctional fatty acid synthase in a transformed Brassica plant cell. [0133]
Plasmid vectors that provide for easy selection, amplification, and transformation of the transgene in prokaryotic and eukaryotic cells include, for example, pUC-derived vectors, pSK-derived vectors, pGEM-derived vectors, pSP-derived vectors, pBS-derived vectors, pFastBac (Invitrogen) for baculovirus expression and pYES2 (Invitrogen) for yeast expression. Additional elements may be present in such vectors, including origins of replication to provide for autonomous replication of the vector, selectable marker genes, preferably encoding antibiotic or herbicide resistance, unique multiple cloning sites providing for multiple sites to insert DNA sequences or genes encoded in the transgene, and sequences that enhance transformation of prokaryotic and eukaryotic cells. One vector that is useful for expression in both plant and prokaryotic cells is the binary Ti plasmid (as disclosed in Schilperoot et al., U.S. Pat. No. 4,940,838), as exemplified by vector pGA582. This binary Ti plasmid vector has been previously characterized by An, [0134] Methods in Enzymology, 153:292 (1987). This binary Ti vector can be replicated in prokaryotic bacteria, such as E. coli and Agrobacterium. The Agrobacterium plasmid vectors can also be used to transfer the transgene to Brassica plant cells. The binary Ti vectors preferably include the nopaline T DNA right and left borders to provide for efficient Brassica plant cell transformation, a selectable marker gene, unique multiple cloning sites in the T border regions, the colE1 replication of origin and a wide host range replicon. The binary Ti vectors carrying a transgene of the present invention can be used to transform both prokaryotic and eukaryotic cells, but is preferably used to transform plant cells. See, for example, Glassman et al., U.S. Pat. No. 5,258,300.
In general, the expression vectors and cassettes of the present invention contain at least a promoter capable of expressing RNA in a Brassica plant cell and a terminator, in addition to a nucleic acid encoding a multifunctional fatty acid synthase. Other elements may also be present in the expression cassettes of the present invention. For example, expression cassettes can also contain enhancers, introns, untranslated leader sequences, cloning sites, matrix attachment regions for silencing the effects of chromosomal control elements, and other elements known to one of skill in the art. [0135]
Nucleic acids encoding fatty acid synthases are operably linked to regulatory elements, such as a promoter, termination signals, and the like. Operably linking a nucleic acid under the regulatory control of a promoter or a regulatory element means positioning the nucleic acid such that the expression of the nucleic acid is controlled by these sequences. In general, promoters are found positioned 5′ (upstream) to the nucleic acid that they control. Thus, in the construction of heterologous promoter/nucleic acid combinations, the promoter is preferably positioned upstream to the nucleic acid and at a distance from the transcription start site of the nucleic acid that the distance between the promoter and the transcription start site approximates the distance observed in the natural setting. As is known in the art, some variation in this distance can be tolerated without loss of promoter function. Similarly, the preferred positioning of a regulatory element with respect to a heterologous nucleic acid placed under its control is the natural position of the regulatory element relative to the structural gene it naturally regulates. Again, as is known in the art, some variation in this distance can be accommodated. [0136]
Expression cassettes have promoters that can regulate gene expression. Promoter regions are typically found in the flanking DNA sequence upstream from coding regions in both prokaryotic and eukaryotic cells. A promoter sequence provides for regulation of transcription of the downstream gene sequence and typically includes from about 50 to about 2,000 nucleotide base pairs. Promoter sequences also contain regulatory sequences, such as enhancer sequences that can influence the level of gene expression. Some isolated promoter sequences can provide for gene expression of heterologous genes, that is, a gene different from the native or homologous gene. Promoter sequences are also known to be strong or weak or inducible. A strong promoter provides for a high level of gene expression, whereas a weak promoter provides for a very low level of gene expression. An inducible promoter is a promoter that provides for turning on and off of gene expression in response to an exogenously added agent or to an environmental or developmental stimulus. Promoters can also provide for tissue specific or developmental regulation. An isolated promoter sequence that is a strong promoter for heterologous genes is advantageous because it provides for a sufficient level of gene expression to allow for easy detection and selection of transformed cells and provides for a high level of gene expression when desired. Transcription initiation regions that are preferentially expressed in seed tissue, and that are undetectable in other Brassica plant parts, are considered desirable for seed oil modifications in order to minimize any disruptive or adverse effects of the gene product. [0137]
Promoters of the present invention will generally include, but are not limited to, promoters that function in bacteria, bacteriophage, plastids, or plant cells. Useful promoters include the globulin promoter (see, for example, Belanger and Kriz, [0138] Genet., 129:863-872, 1991), gamma zein Z27 promoter (see, for example, U.S. Ser. No. 08/763,705; also Lopes et al., Mol Gen Genet., 247:603-613, 1995), L3 oleosin promoter (U.S. Pat. No. 6,433,252), USP promoter and 7Sα promoter (U.S. Ser. No. 10/235,618), 7Sα′ promoter (see, for example, Beachy et al., EMBO J., 4:3047, 1985; Schuler et al., Nucleic Acid Res., 10(24):8225-8244, 1982), CaMV ³⁵S promoter (Odell et al., Nature, 313:810, 1985), the CaMV 19S (Lawton et al., Plant Mol. Biol., 9:31F, 1987), nos (Ebert et al., Proc. Natl. Acad. Sci. (U.S.A.), 84:5745, 1987), Adh (Walker et al., Proc. Natl. Acad. Sci. (U.S.A.), 84:6624, 1987), sucrose synthase (Yang et al., Proc. Natl. Acad. Sci. (U.S.A.), 87:4144, 1990), tubulin, actin (Wang et al., Mol. Cell. Biol., 12:3399, 1992), cab (Sullivan et al., Mol. Gen. Genet., 215:431, 1989), PEPCase promoter (Hudspeth et al., Plant Mol. Biol., 12:579, 1989), or those associated with the R gene complex (Chandler et al., The Plant Cell, 1: 1175, 1989). Other useful promoters include the Figwort Mosaic Virus (FMV) promoter (Richins et al., Nucleic Acids Res., 20:8451, 1987), arcelin, tomato E8, patatin, ubiquitin, mannopine synthase (mas), soybean seed protein glycinin (Gly), soybean vegetative storage protein (vsp), bacteriophage SP6, T3, and T7 promoters.
Indeed, in a preferred embodiment, the promoter used is a seed-specific promoter. Examples of seed regulated genes and transcriptional regions are disclosed in U.S. Pat. Nos. 5,420,034; 5,608,152; and 5,530,194. Examples of such promoters include the 5′ regulatory regions from such genes as napin (Kridl et al., [0139] Seed Sci. Res., 1:209-219, 1991), phaseolin (Bustos et al., Plant Cell, 1(9):839-853, 1989), soybean trypsin inhibitor (Riggs et al., Plant Cell, 1(6):609-621, 1989), ACP (Baerson et al., Plant Mol. Biol., 22(2):255-267, 1993), stearoyl-ACP desaturase (Slocombe et al., Plant Physiol., 104(4):167-176, 1994), soybean a′ subunit of p-conglycinin (Chen et al., Proc. Natl. Acad. Sci., 83:8560-8564, 1986), Lesquerella hydroxylase promoter (described in Broun et al., Plant Journal, 12(2):201-210, 1998; U.S. Pat. No. 5,965,793), delta 12 desaturase and oleosin (Hong et al., Plant Mol. Biol., 34(3):549-555, 1997). Further examples include the promoter for β-conglycinin (Chen et al., Dev. Genet., 10:112-122, 1989), the GL2 promoter (Szymanski et al., Development, 125:1161-1171, 1998), the tt2 promoter (Nesi et al., The Plant Cell, 13:2099-114, 2001), the LDOX promoter (Pelletier et al., Plant Physiology, 113:1437-1445, 1997), the CPC promoter (Wada et al., Science, 277:1113-1116, 1997).
Plastid promoters can also be used. Most plastid genes contain a promoter for the multi-subunit plastid-encoded RNA polymerase (PEP) as well as the single-subunit nuclear-encoded RNA polymerase. A consensus sequence for the nuclear-encoded polymerase (NEP) promoters and listing of specific promoter sequences for several native plastid genes can be found in Hajdukiewicz et al., [0140] EMBO J., 16:4041-4048 (1997), which is hereby in its entirety incorporated by reference.
Examples of plastid promoters that can be used include the [0141] Zea mays plastid RRN (ZMRRN) promoter. The ZMRRN promoter can drive expression of a gene when the Arabidopsis thaliana plastid RNA polymerase is present. Similar promoters that can be used in the present invention are the Glycine max plastid RRN(SOYRRN) and the Nicotiana tabacum plastid RRN (NTRRN) promoters. All three promoters can be recognized by the Arabidopsis plastid RNA polymerase. The general features of RRN promoters are described by Hajdukiewicz et al., supra, and U.S. Pat. No. 6,218,145.
Moreover, transcription enhancers or duplications of enhancers can be used to increase expression from a particular promoter. Examples of such enhancers include, but are not limited to, elements from the CaMV [0142] ³⁵S promoter and octopine synthase genes (Last et al., U.S. Pat. No. 5,290,924). As the DNA sequence between the transcription initiation site and the start of the coding sequence, i.e., the untranslated leader sequence, can influence gene expression, one may also wish to employ a particular leader sequence. Any leader sequence available to one of skill in the art may be employed. Preferred leader sequences direct optimum levels of expression of the attached gene, for example, by increasing or maintaining mRNA stability and/or by preventing inappropriate initiation of translation (Joshi, Nucl. Acid Res., 15:6643, 1987). The choice of such sequences is at the discretion of those of skill in the art. Sequences that are derived from genes that are highly expressed in Brassica in particular are contemplated.
An inducible promoter can be turned on or off by an exogenously added agent so that expression of an operably linked nucleic acid is also turned on or off. For example, a bacterial promoter, such as the P[0143] _tac, promoter can be induced to varying levels of gene expression depending on the level of isothiopropylgalactoside added to the transformed bacterial cells. It may also be preferable to combine the nucleic acid encoding the polypeptide of interest with a promoter that provides tissue specific expression or developmentally regulated gene expression in plants.
Expression cassettes of the present invention will also include a sequence near the 3′ end of the cassette that acts as a signal to terminate transcription from a heterologous nucleic acid and that directs polyadenylation of the resultant mRNA. Some 3′ elements that can act as termination signals include those from the nopaline synthase gene of [0144] Agrobacterium tumefaciens (Bevan et al., Nucl. Acid Res., 11:369, 1983), a napin 3′ untranslated region (Kridl et al., Seed Sci Res., 1:209-219, 1991), a globulin 3′ untranslated region (Belanger and Kriz, Genetics, 129:863-872, 1991), or one from a zein gene, such as Z27 (Lopes et al., Mol Gen Genet., 247:603-613, 1995). Other 3′ elements known by one of skill in the art also can be used in the vectors of the present invention.
Regulatory elements, such as Adh intron 1 (Callis et al., [0145] Genes Develop., 1: 1183, 1987), a rice actin intron (McElroy et al., Mol. Gen. Genet., 231(1):150-160, 1991), sucrose synthase intron (Vasil et al., Plant Physiol., 91:5175, 1989), the maize HSP70 intron (Rochester et al., EMBO J., 5:451-458, 1986), or TMV omega element (Gallie et al., The Plant Cell, 1:301, 1989) may further be included where desired. These 3′ nontranslated regulatory sequences can be obtained as described in An, Methods in Enzymology, 153:292 (1987) or are already present in plasmids available from commercial sources, such as Clontech, Palo Alto, Calif. The 3′ nontranslated regulatory sequences can be operably linked to the 3′ terminus of any heterologous nucleic acid to be expressed by the expression cassettes contained within the present vectors. Other such regulatory elements useful in the practice of the present invention are known by one of skill in the art and can also be placed in the vectors of the present invention.
The vectors of the present invention, as well as the coding regions claimed herein, can be optimized for expression in Brassica plants by having one or more codons replaced by other codons encoding the same amino acids so that the polypeptide is optimally translated by the translation machinery of the Brassica plant species in which the vector is used. [0146]
Selectable Markers: Selectable marker genes or reporter genes are also useful in the present invention. Such genes can impart a distinct phenotype to cells expressing the marker gene and thus allow such transformed cells to be distinguished from cells that do not have the marker. Selectable marker genes confer a trait that one can ‘select’ for by chemical means, i.e., through the use of a selective agent (e.g., a herbicide, antibiotic, or the like). Reporter genes or screenable genes, confer a trait that one can identify through observation or testing, i.e., by ‘screening’ (e.g., the R-locus trait). Of course, many examples of suitable marker genes are known to the art and can be employed in the practice of the present invention. [0147]
Possible selectable markers for use in connection with the present invention include, but are not limited to, a neo gene (Potrykus et al., [0148] Mol. Gen. Genet., 199:183, 1985) which codes for kanamycin resistance and can be selected for by applying kanamycin, a kanamycin analog such as geneticin (Sigma Chemical Company, St. Louis, Mo.), and the like; a bar gene that codes for bialaphos resistance; a gene that encodes an altered EPSP synthase protein (Hinchee et al., Biotech., 6:915, 1988) thus conferring glyphosate resistance; a nitrilase gene, such as bxn from Klebsiella ozaenae, which confers resistance to bromoxynil (Stalker et al., Science, 242:419, 1988); a mutant acetolactate synthase gene (ALS) that confers resistance to imidazolinone, sulfonylurea, or other ALS-inhibiting chemicals (EP 154 204A1, 1985); a methotrexate-resistant DHFR gene (Thillet et al., J. Biol. Chem., 263:12500, 1988); a dalapon dehalogenase gene that confers resistance to the herbicide dalapon. Where a mutant EPSP synthase gene is employed, additional benefit may be realized through the incorporation of a suitable plastid transit peptide (CTP).
An illustrative embodiment of a selectable marker gene capable of being used in systems to select transformants is the genes that encode the enzyme phosphinothricin acetyltransferase, such as the bar gene from Streptomyces hygroscopicus or the pat gene from Streptomyces viridochromogenes (U.S. Pat. No. 5,550,318, which is incorporated by reference herein). The enzyme phosphinothricin acetyl transferase (PAT) inactivates the active ingredient in the herbicide bialaphos, phosphinothricin that inhibits glutamine synthetase, (Murakami et al., [0149] Mol. Gen. Genet., 205:42, 1986; Twell et al., Plant Physiol., 91:1270, 1989) causing rapid accumulation of ammonia and cell death.
Screenable markers that may be employed include, but are not limited to, a P-glucuronidase or uidA gene (GUS), which encodes an enzyme for which various chromogenic substrates are known; an R-locus gene, which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al., [0150] Chromosome Structure and Function, 263-282, 1988); a P-lactamase gene (Sutcliffe, Proc. Natl. Acad. Sci. (U.S.A.), 75:3737, 1978), which encodes an enzyme for which various chromogenic substrates are known (e.g., PADAC, a chromogenic cephalosporin); a xylE gene (Zukowsky et al., Proc. Natl. Acad. Sci. (U.S.A.), 80:1101, 1983) that encodes a catechol dioxygenase that can convert chromogenic catechols; an α-amylase gene (Ikuta et al., Biotech., 8:241, 1990); a tyrosinase gene (Katz et al., J. Gen. Microbiol., 129:2703, 1983) that encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which in turn condenses to form the easily detectable compound melanin; a β-galactosidase gene, which encodes an enzyme for which there are chromogenic substrates; a luciferase (lux) gene (Ow et al., Science, 234:856, 1986), which allows for bioluminescence detection; or an aequorin gene (Prasher et al., Biochem. Biophys. Res. Comm., 126:1259, 1985), which may be employed in calcium-sensitive bioluminescence detection, or a green fluorescent protein gene (Niedz et al., Plant Cell Reports, 14:403, 1995). The presence of the lux gene in transformed cells may be detected using, for example, X-ray film, scintillation counting, fluorescent spectrophotometry, low-light video cameras, photon-counting cameras, or multiwell luminometry. It is also envisioned that this system may be developed for populational screening for bioluminescence, such as on tissue culture plates, or even for whole plant screening.
Transit Peptides: Additionally, transgenes may be constructed and employed to provide targeting of the gene product to an intracellular compartment within plant cells or in directing a protein to the extracellular environment. This will generally be achieved by joining a DNA sequence encoding a transit or signal peptide sequence to the coding sequence of a particular gene. The resultant transit or signal peptide will transport the protein to a particular intracellular, or extracellular destination, respectively, and may then be post-translationally removed. Transit or signal peptides act by facilitating the transport of proteins through intracellular membranes, e.g., vacuole, vesicle, plastid, and mitochondrial membranes, whereas signal peptides direct proteins through the extracellular membrane. By facilitating transport of the protein into compartments inside or outside the cell, these sequences may increase the accumulation of gene product. [0151]
An example of such a use concerns the direction of a fatty acid synthase to a particular organelle, such as to a plastid rather than to the cytoplasm. This is exemplified by the use of the Arabidopsis SSU1A transit peptide that confers plastid-specific targeting of proteins. Alternatively, the transgene can comprise a plastid transit peptide-encoding DNA sequence or a DNA sequence encoding the rbcS (RuBISCO) transit peptide operably linked between a promoter and the DNA sequence encoding a fatty acid synthase (for a review of plastid targeting peptides, see Heijne et al., [0152] Eur. J. Biochem., 180:535, 1989; Keegstra et al., Ann. Rev. Plant Physiol. Plant Mol. Biol., 40:471, 1989). If the transgene is to be introduced into a plant cell, the transgene can also contain plant transcriptional termination and polyadenylation signals and translational signals linked to the 3′ terminus of a plant fatty acid synthase gene.
A heterologous plastid transit peptide can be linked to a multifunctional fatty acid synthase gene. A plastid transit peptide is typically 40 to 70 amino acids in length and functions post-translationally to direct a protein to the plastid. The transit peptide is cleaved either during or just after import into the plastid to yield the mature protein. [0153]
Heterologous plastid transit peptide encoding sequences can be obtained from a variety of plant nuclear genes, so long as the products of the genes are expressed as preproteins comprising an amino terminal transit peptide and transported into plastid. Examples of plant gene products known to include such transit peptide sequences include, but are not limited to, the small subunit of ribulose biphosphate carboxylase, chlorophyll a/b binding protein, plastid ribosomal proteins encoded by nuclear genes, certain heat shock proteins, amino acid biosynthetic enzymes, such as acetolactate acid synthase, 3-enolpyruvylphosphoshikimate synthase, dihydrodipicolinate synthase, fatty acid synthase, and the like. In some instances, a plastid transport protein already may be encoded in the fatty acid synthase gene of interest, in which case there may be no need to add such plastid transit sequences. Alternatively, the DNA fragment coding for the transit peptide may be chemically synthesized either wholly or in part from the known sequences of transit peptides such as those listed above. [0154]
Regardless of the source of the DNA fragment coding for the transit peptide, it should include a translation initiation codon, for example, an ATG codon, and be expressed as an amino acid sequence that is recognized by and will function properly in plastids of the host plant. Attention should also be given to the amino acid sequence at the junction between the transit peptide and the fatty acid synthase enzyme, where it is cleaved to yield the mature enzyme. Certain conserved amino acid sequences have been identified and may serve as a guideline. Precise fusion of the transit peptide coding sequence with the fatty acid synthase coding sequence may require manipulation of one or both DNA sequences to introduce, for example, a convenient restriction site. This may be accomplished by methods including site-directed mutagenesis, insertion of chemically synthesized oligonucleotide linkers, and the like. [0155]
Precise fusion of the nucleic acids encoding the plastid transport protein may not be necessary so long as the coding sequence of the plastid transport protein is in-frame with that of the fatty acid synthase. For example, additional peptidyl or amino acids can often be included without adversely affecting the expression or localization of the protein of interest. [0156]
Once obtained, and when desired, the plastid transit peptide sequence can be appropriately linked to the promoter and a fatty acid synthase coding region in a transgene using standard methods. A plasmid containing a promoter functional in plant cells and having multiple cloning sites downstream can be constructed or obtained from commercial sources. The plastid transit peptide sequence can be inserted downstream from the promoter using restriction enzymes. A fatty acid synthase coding region can then be translationally fused or inserted immediately downstream from and in frame with the 3′ terminus of the plastid transit peptide sequence. Hence, the plastid transit peptide is preferably linked to the amino terminus of the fatty acid synthase. Once formed, the transgene can be subcloned into other plasmids or vectors. [0157]
In addition to nuclear plant transformation, the present invention also extends to direct transformation of the plastid genome of Brassica plants. Hence, targeting of the gene product to an intracellular compartment within plant cells may also be achieved by direct delivery of a gene to the intracellular compartment. In some embodiments, direct transformation of plastid genome may provide additional benefits over nuclear transformation. For example, direct plastid transformation of fatty acid synthase eliminates the requirement for a plastid targeting peptide and post-translational transport and processing of the pre-protein derived from the corresponding nuclear transformants. Plastid transformation of plants has been described by Maliga, [0158] Current Opinion in Plant Biology, 5:164-172 (2002); Heifetz, Biochimie, 82:655-666 (2000); Bock, J. Mol. Biol., 312:425-438 (2001); and Daniell et al., Trends in Plant Science, 7:84-91 (2002), and references cited therein.
After constructing a transgene containing a multifunctional fatty acid synthase, the expression vector or cassette can then be introduced into a Brassica plant cell. Depending on the type of plant cell, the level of gene expression, and the activity of the enzyme encoded by the gene, introduction of DNA encoding a multifunctional fatty acid synthase into the plant cell can lead to increased oil content in Brassica plant tissues. [0159]
Plant Transformation: There are many methods for introducing transforming nucleic acid molecules into plant cells. Suitable methods are believed to include virtually any method by which nucleic acid molecules may be introduced into a cell, such as by Agrobacterium infection or direct delivery of nucleic acid molecules, such as, for example, by PEG-mediated transformation, by electroporation or by acceleration of DNA coated particles, and the like. (Potrykus, [0160] Ann. Rev. Plant Physiol. Plant Mol. Biol., 42:205-225, 1991; Vasil, Plant Mol. Biol., 25:925-937, 1994). For example, electroporation has been used to transform maize protoplasts (Fromm et al., Nature, 312:791-793, 1986).
Other vector systems suitable for introducing transforming DNA into a host plant cell include but are not limited to binary artificial chromosome (BIBAC) vectors (Hamilton et al., [0161] Gene, 200:107-116, 1997); and transfection with RNA viral vectors (Della-Cioppa et al., Ann. N.Y. Acad. Sci., (1996), 792 (Engineering Plants for Commercial Products and Applications, 57-61)). Additional vector systems also include plant selectable YAC vectors, such as those described in Mullen et al., Molecular Breeding, 4:449-457 (1988).
Technology for introduction of DNA into cells is well known by one of skill in the art. Four general methods for delivering a gene into cells have been described: (1) chemical methods (Graham and van der Eb, Virology, 54:536-539, 1973); (2) physical methods, such as microinjection (Capecchi, [0162] Cell, 22:479-488, 1980), electroporation (Wong and Neumann, Biochem. Biophys. Res. Commun., 107:584-587, 1982; Fromm et al., Proc. Natl. Acad. Sci. (U.S.A.), 82:5824-5828, 1985; U.S. Pat. No. 5,384,253); the gene gun (Johnston and Tang, Methods Cell Biol., 43:353-365, 1994); and vacuum infiltration (Bechtold et al., C. R. Acad. Sci. Paris, Life Sci., 316:1194-1199, 1993); (3) viral vectors (Clapp, Clin. Perinatol., 20:155-168, 1993; Lu et al., J. Exp. Med., 178:2089-2096, 1993; Eglitis and Anderson, Biotechniques, 6:608-614, 1988); and (4) receptor-mediated mechanisms (Curiel et al., Hum. Gen. Ther., 3:147-154, 1992; Wagner et al., Proc. Natl. Acad. Sci. (U.S.A.), 89:6099-6103, 1992).
Acceleration methods that may be used include, for example, microprojectile bombardment and the like. One example of a method for delivering transforming nucleic acid molecules into plant cells is microprojectile bombardment. This method has been reviewed by Yang and Christou (eds.), [0163] Particle Bombardment Technology for Gene Transfer, Oxford Press, Oxford, England (1994). Non-biological particles (microprojectiles) may be coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, gold, platinum, and the like.
A particular advantage of microprojectile bombardment, in addition to it being an effective means of reproducibly transforming monocots, is that neither the isolation of protoplasts (Cristou et al., [0164] Plant Physiol., 87:671-674, 1988) nor the susceptibility to Agrobacterium infection is required. An illustrative embodiment of a method for delivering DNA into maize cells by acceleration is a biolistics α-particle delivery system, which can be used to propel particles coated with DNA through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with maize cells cultured in suspension. Gordon-Kamm et al., describes the basic procedure for coating tungsten particles with DNA (Gordon-Kamm et al., Plant Cell, 2:603-618, 1990). The screen disperses the tungsten nucleic acid particles so that they are not delivered to the recipient cells in large aggregates. A particle delivery system suitable for use with the present invention is the helium acceleration PDS-1000/He gun, which is available from Bio-Rad Laboratories (Bio-Rad, Hercules, Calif.) (also, see, Sanford et al., Technique, 3:3-16, 1991).
For the bombardment, cells in suspension may be concentrated on filters. Filters containing the cells to be bombarded are positioned at an appropriate distance below the microprojectile stopping plate. If desired, one or more screens are also positioned between the gun and the cells to be bombarded. [0165]
Alternatively, immature embryos or other target cells may be arranged on solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the microprojectile stopping plate. If desired, one or more screens are also positioned between the acceleration device and the cells to be bombarded. Through the use of techniques set forth herein one may obtain 1000 or more loci of cells transiently expressing a marker gene. The number of cells in a focus that express the exogenous gene product 48 hours post-bombardment often ranges from 1 to 10, and average 1 to 3. [0166]
In bombardment transformation, one may optimize the pre-bombardment culturing conditions and the bombardment parameters to yield the maximum numbers of stable transformants. Both the physical and biological parameters for bombardment are important in this technology. Physical factors are those that involve manipulating the DNA/microprojectile precipitate or those that affect the flight and velocity of the microprojectiles. Biological factors include all steps involved in manipulation of cells before and immediately after bombardment, the osmotic adjustment of target cells to help alleviate the trauma associated with bombardment and, also, the nature of the transforming DNA, such as linearized DNA or intact supercoiled plasmids. It is believed that pre-bombardment manipulations are especially important for successful transformation of immature embryos. [0167]
In another alternative embodiment, plastids can be stably transformed. Methods disclosed for plastid transformation in higher plants include the particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination (Svab et al., [0168] Proc. Natl. Acad. Sci. (U.S.A.), 87:8526-8530, 1990; Svab and Maliga, Proc. Natl. Acad. Sci. (U.S.A.), 90:913-917, 1993; Staub and Maliga, EMBO J., 12:601-606, 1993; U.S. Pat. Nos. 5,451,513 and 5,545,818).
Accordingly, it is contemplated that one may wish to adjust various aspects of the bombardment parameters in small scale studies to fully optimize the conditions. One may particularly wish to adjust physical parameters such as gap distance, flight distance, tissue distance, and helium pressure. One may also minimize the trauma reduction factors by modifying conditions that influence the physiological state of the recipient cells and which may therefore influence transformation and integration efficiencies. For example, the osmotic state, tissue hydration, and the subculture stage or cell cycle of the recipient cells may be adjusted for optimum transformation. The execution of other routine adjustments will be known by one of skill in the art in light of the present invention. [0169]
Agrobacterium-mediated transfer is a widely applicable system for introducing genes into plant cells because the DNA can be introduced into whole plant tissues, thereby bypassing the need for regeneration of an intact plant from a protoplast. The use of Agrobacterium-mediated plant integrating vectors to introduce DNA into plant cells is well known in the art. See, for example the methods described by Fraley et al., [0170] BioTechnology, 3:629-635 (1985) and Rogers et al., Methods Enzymol., 153:253-277 (1987). Further, the integration of the Ti-DNA is a relatively precise process resulting in few rearrangements. The region of DNA to be transferred is defined by the border sequences and intervening DNA is usually inserted into the plant genome as described (Spielmann et al., Mol. Gen. Genet., 205:34, 1986).
Modern Agrobacterium transformation vectors are capable of replication in [0171] E. coli as well as Agrobacterium, allowing for convenient manipulations as described (Klee et al., In: Plant DNA Infectious Agents, Hohn and Schell (eds.), Springer-Verlag, NY, pp. 179-203, 1985). Moreover, technological advances in vectors for Agrobacterium-mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate construction of vectors capable of expressing various polypeptide coding genes. The vectors described have convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes and are suitable for present purposes (Rogers et al., Methods Enzymol., 153:253-277, 1987). In addition, Agrobacterium containing both armed and disarmed Ti genes can be used for the transformations. In those plant strains where Agrobacterium-mediated transformation is efficient, it is the method of choice because of the facile and defined nature of the gene transfer.
A transgenic plant formed using Agrobacterium transformation methods typically contains a single gene on one chromosome. Such transgenic plants can be referred to as being heterozygous for the added gene. More preferred is a transgenic plant that is homozygous for the added structural gene; i.e., a transgenic plant that contains 2 added genes, one gene at the same locus on each chromosome of a chromosome pair. A homozygous transgenic plant can be obtained by sexually mating (selfing) an independent segregant, transgenic plant that contains a single added gene, germinating some of the seed produced and analyzing the resulting plants produced for the gene of interest. [0172]
It is also to be understood that two different transgenic plants can also be mated to produce offspring that contain two independently segregating, exogenous genes. Selfing of appropriate progeny can produce plants that are homozygous for both added, exogenous genes that encode a polypeptide of interest. Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated, as is vegetative propagation. [0173]
Transformation of plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments (see, for example, Potrykus et al., [0174] Mol. Gen. Genet., 205:193-200, 1986; Lorz et al., Mol. Gen. Genet., 199:178, 1985; Fromm et al., Nature, 319:791, 1986; Uchimiya et al., Mol. Gen. Genet., 204:204, 1986; Marcotte et al., Nature, 335:454-457, 1988).
Application of these systems to different plant strains depends upon the ability to regenerate that particular plant strain from protoplasts. Illustrative methods for the regeneration of cereals from protoplasts are described (Fujimura et al., [0175] Plant Tissue Culture Letters, 2:74, 1985; Toriyama et al., Theor. Appl. Genet., 205:34, 1986; Yamada et al., Plant Cell Rep., 4:85, 1986; Abdullah et al., Biotechnology, 4:1087, 1986).
To transform plant strains that cannot be successfully regenerated from protoplasts, other ways to introduce DNA into intact cells or tissues can be utilized. For example, regeneration of cereals from immature embryos or explants can be effected as described (Vasil, [0176] Biotechnology, 6:397, 1988). In addition, “particle gun” or high-velocity microprojectile technology can be utilized (Vasil et al., Bio/Technology, 10:667, 1992).
Using the latter technology, DNA is carried through the cell wall and into the cytoplasm on the surface of small metal particles as described (Klein et al., [0177] Nature, 328:70, 1987; Klein et al., Proc. Natl. Acad. Sci. (U.S.A.), 85:8502-8505, 1988; McCabe et al., Bio/Technology, 6:923, 1988). The metal particles penetrate through several layers of cells and thus allow the transformation of cells within tissue explants.
Other methods of cell transformation can also be used and include but are not limited to introduction of DNA into plants by direct DNA transfer into pollen (Hess et al., [0178] Intern Rev. Cytol., 107:367, 1987; Luo et al., Plant Mol. Biol. Reporter, 6:165, 1988), by direct injection of DNA into reproductive organs of a plant (Pena et al., Nature, 325:274, 1987), or by direct injection of DNA into the cells of immature embryos followed by the rehydration of desiccated embryos (Neuhaus et al., Theor. Appl. Genet., 75:30, 1987).
The regeneration, development, and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art (Weissbach and Weissbach, In: [0179] Methods for Plant Molecular Biology, Academic Press, San Diego, Calif., 1988). This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.
The development or regeneration of plants containing the foreign, exogenous gene that encodes a protein of interest is well known in the art. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants. A transgenic plant of the present invention containing a desired polypeptide is cultivated using methods well known to one skilled in the art. [0180]
There are a variety of methods for the regeneration of plants from plant tissue. The particular method of regeneration will depend on the starting plant tissue and the particular plant species to be regenerated. [0181]
Methods for transforming dicots, primarily by use of [0182] Agrobacterium tumefaciens and obtaining transgenic plants have been published for cotton (U.S. Pat. Nos. 5,004,863; 5,159,135; and 5,518,908); soybean (U.S. Pat. Nos. 6,384,301; 5,569,834; and 5,416,011; McCabe et al., Biotechnology, 6:923, 1988; Christou et al., Plant Physiol., 87:671-674, 1988); Brassica (U.S. Pat. No. 5,463,174); peanut (Cheng et al., Plant Cell Rep., 15:653-657, 1996; McKently et al., Plant Cell Rep., 14:699-703, 1995); papaya; pea (Grant et al., Plant Cell Rep., 15:254-258, 1995); and Arabidopsis thaliana (Bechtold et al., C. R. Acad. Sci. Paris, Life Sci., 316:1194-1199, 1993). The latter method for transforming Arabidopsis thaliana is commonly called “dipping” or vacuum infiltration or germplasm transformation.
Transformation of monocotyledons using electroporation, particle bombardment and Agrobacterium have also been reported. Transformation and plant regeneration have been achieved in asparagus (Bytebier et al., [0183] Proc. Natl. Acad. Sci. (U.S.A.), 84:5354, 1987); barley (Wan and Lemaux, Plant Physiol., 104:37, 1994); maize (Rhodes et al., Science, 240:204, 1988; Gordon-Kamm et al., Plant Cell, 2:603-618, 1990; Fromm et al., Bio/Technology, 8:833, 1990; Koziel et al., Bio/Technology, 11: 194, 1993; Armstrong et al., Crop Science, 35:550-557, 1995); oat (Somers et al., Bio/Technology, 10: 1589, 1992); orchard grass (Horn et al., Plant Cell Rep., 7:469, 1988); rice (Toriyama et al., Theor Appl. Genet., 205:34, 1986; Part et al., Plant Mol. Biol., 32:1135-1148, 1996; Abedinia et al., Aust. J. Plant Physiol., 24:133-141, 1997; Zhang and Wu, Theor. Appl. Genet., 76:835, 1988; Zhang et al., Plant Cell Rep., 7:379, 1988; Battraw and Hall, Plant Sci., 86:191-202, 1992; Christou et al., Bioffechnology, 9:957, 1991); rye (DelaPena et al., Nature, 325:274, 1987); sugarcane (Bower and Birch, Plant J., 2:409, 1992); tall fescue (Wang et al., Bio/Technology, 10:691, 1992); and wheat (Vasil et al., Bio/Technology, 10:667, 1992; U.S. Pat. No. 5,631,152).
Assays for gene expression based on the transient expression of cloned nucleic acid constructs have been developed by introducing the nucleic acid molecules into plant cells by polyethylene glycol treatment, electroporation, or particle bombardment (Marcotte et al., [0184] Nature, 335:454-457, 1988; Marcotte et al., Plant Cell, 1:523-532, 1989; McCarty et al., Cell, 66:895-905, 1991; Hattori et al., Genes Dev., 6:609-618, 1992; Goff et al., EMBO J., 9:2517-2522, 1990). Transient expression systems may be used to functionally dissect gene constructs (see generally, Maliga et al., Methods in Plant Molecular Biology, Cold Spring Harbor Press, 1995).
Any of the nucleic acid molecules of the present invention may be introduced into a plant cell in a permanent or transient manner in combination with other genetic elements such as vectors, promoters, enhancers, etc. Further, any of the nucleic acid molecules of the present invention may be introduced into a plant cell in a manner that allows for expression or overexpression of the protein or fragment thereof encoded by the nucleic acid molecule. [0185]
It is also to be understood that 2 different transgenic Brassica plants can also be mated to produce offspring that contain 2 independently segregating added, exogenous genes. Selfing of appropriate progeny can produce plants that are homozygous for both added, exogenous genes that encode a polypeptide of interest. Backcrossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated, as is vegetative propagation. [0186]
Transgenic Brassica plants may find use in the commercial manufacture of proteins or other molecules, where the molecule of interest is extracted or purified from plant parts, seeds, and the like. Cells or tissue from the plants may also be cultured, grown in vitro, or fermented to manufacture such molecules. [0187]
The transgenic Brassica plants may also be used in commercial breeding programs, or may be crossed or bred to plants of related crop species. Improvements encoded by the recombinant DNA may be transferred, e.g., from cells of one species to cells of other species, e.g., by protoplast fusion. [0188]
The present invention also provides for a method of stably expressing a fatty acid synthase of interest in a Brassica plant, which includes, contacting the plant cell with a vector of the present invention that has a selectable marker gene and a nucleic acid encoding the fatty acid synthase of interest, under conditions effective to transform the plant cell. A promoter within the expression cassette can be any of the promoters provided herein, for example, a constitutive promoter, an inducible promoter, a tissue-specific promoter or a seed specific promoter. Such promoters can provide expression of an encoded fatty acid synthase at a desired time, or at a desired developmental stage, or in a desired tissue. [0189]
The present invention also provides for a method of stably expressing a fatty acid synthase of interest in a plant, which includes, contacting the plant cell with a vector of the present invention that has a nucleic acid encoding the fatty acid synthase of interest, under conditions effective to transfer and integrate the vector into the nuclear genome of the cell. The vector can also include a selectable marker gene. When using the vector with [0190] Agrobacterium tumefaciens, the vector can have an Agrobacterium tumefaciens origin of replication.
In another embodiment, the present invention provides a method of producing a Brassica oil, comprising the steps of: a) growing an oilseed Brassica plant, the genome of which contains a nucleic acid molecule encoding a multifunctional fatty acid synthase, to produce oil-containing seeds; and b) extracting oil from the seeds. In another embodiment, the present invention provides a method of producing a Brassica oil, comprising the steps of: [0191]
a) growing an oilseed Brassica plant, the genome of which contains a nucleic acid molecule encoding a phosphopantetheine:protein transferase, to produce oil-containing seeds; and b) extracting oil from the seeds. [0192]
Plants: Plants for use with the vectors of the present invention include Brassica sp., particularly those Brassica species useful as sources of seed oil (e.g., [0193] B. napus, B. rapa, B. juncea).
The following examples are provided to illustrate the present invention and are not intended to limit the present invention in any way. [0194]

EXAMPLE 1

This example describes the isolation of the fasA and ppt1 genes from Brevibacterium ammoniagenes. [0195]
Genomic DNA was isolated from B. ammoniagenes (ATCC 5871) using standard methodologies. A genomic library was prepared by partially digesting B. ammoniagenes genomic DNA with the restriction enzyme Sau3A, isolating DNA fragments ranging from 30-42 kb in size and generating the library using the [0196] SuperCos 1 Cosmid Vector kit from Stratagene, Inc. (La Jolla, Calif.). The genomic library was screened by hybridization and washing under stringent conditions with a 2P-labelled 1.1 kb fasA PCR fragment generated from isolated genomic DNA using the following PCR primers:

14713 (forward):

5′-CCAGCTCAACGATGAAGTAG-3′ [SEQ ID NO: 5]

and

14714 (reverse):

5′-TCGATGATCTGGTCTACTTC-3′ [SEQ ID NO: 6]
Prehybridization was in a solution of 40% formamide, 5×SSC, 50 mM sodium phosphate, pH 7.0, 5× Denhardt's, 0.1% SDS, 5 mM EDTA, 0.1 μg/ml salmon sperm DNA, and 5% Dextran sulfate for 2 hrs at 42° C. and hybridization was in the same solution as described overnight at 42° C. The filters were rinsed briefly in 0.1×SSC, 0.1% SDS at RT and then washed 2 times for 20 min each in 0.1×SSC, 0.1% SDS at 50° C. FasA-containing clones were identified by autoradiography and restriction mapping. Selected cosmid clones were analyzed in more detail and one clone was confirmed to have the full-length fasA gene by restriction mapping and comparison with the restriction sites in the published sequence (Stuible et al., [0197] J. Bacteriol., 178:4787-4793, 1993).
The full-length fasA gene was assembled so as to introduce convenient flanking restriction sites for sub-cloning by using the following basic steps: a) PCR amplification of the 5′ and 3′ ends; b) assembling the 5′ and 3′ ends of the gene together by an overlapping PCR strategy resulting in deletion of the fasA sequence between the internal MfeI and XhoI sites; c) cloning the “5′-3′ fused” PCR fragment; d) insertion of the 8166 bp fasA MfeI/XhoI fragment between the MfeI and XhoI sites in the “5′-3′ fused” PCR fragment so as to re-generate the full-length fasA gene with convenient flanking cloning sites. The details for each of these steps are outlined below. [0198]
A 5′ 280 bp fasA PCR fragment was generated using the following primers: [0199]
16393 (forward) [0200]

5′- TCTAGATGCATAGTTAACATGTCGTTGACCCCCTTGC -3′ [SEQ ID NO: 9]

and

14873 (reverse)

5′-GGTACGCGTCATATTCCTTG -3′ [SEQ ID NO: 10]
The forward primer, 16393, introduced XbaI, NsiI, HpaI, and PciI flanking restriction sites. [0201]
A 3′ 946 bp fasA PCR fragment was generated using the following primers: [0202]
16385 (forward) [0203]

5′-CAAGGAATATGACGCGTACCCTCGAGGCAGAAGGCGGCGG- 3′ [SEQ ID NO: 11]

and

16394 (reverse)

5′-ATGCATGTTAACATGTCTACTTTGTCCTACTTCGCCG-3′ [SEQ ID NO: 12]
The reverse primer, 16394, introduced 3′ flanking NsiI, HpaI, and PciI restriction sites. The forward primer, 16385, contained 20 bp of sequence matching the 3′-end of the 5′ 280 bp restriction fragment described above to allow the 2 fragments to anneal together. The 5′ 280 bp fasA PCR fragment and the 3′ 946 bp fasA PCR fragment were fused together by annealing the 2 fragments and PCR amplifying the full length (1206 bp) overlapped fragment using the external primers 16393 (forward) and 16394 (reverse). The 1206 bp 5′-3′-fused PCR fragment was cloned into pCR-Blunt II-TOPO (Invitrogen Corporation, Carlsbad, Calif.) and the correct DNA sequence was confirmed by sequencing. The 1206 bp 5′-3′-fused PCR fragment was then sub-cloned as an SpeI/XbaI fragment into a Bluescript pBC KS+(Stratagene Inc., La Jolla, Calif.) vector which contained a modified multiple cloning sequence (pCGN3686). The full-length fasA gene was then obtained by ligation of the 3505 bp MluI/MluI and 4516 bp MluI/XhoI internal fasA fragments isolated from the full-length cosmid clone between the MluI and XhoI sites in the 5′-3′ fused PCR fragment to make pMON70058 (FIG. 1). [0204]
The complete double-stranded sequence of the full-length fasA gene open reading frame in pMON70058 was determined using a Perkin Elmer ABI 377 DNA sequencer (SEQ ID NO: 1). The corresponding protein sequence (SEQ ID NO: 2) was predicted based on standard genetic code using the program Omiga (Accelrys, Inc., Cambridge, UK) and compared with the published fasA sequence (FIG. 2). Alignment of both the nucleic acid and predicted amino acid sequences to the published sequences (Stuible et al., [0205] J. Bacteriol., 178:4787, 1996) revealed a number of differences both at the DNA and protein levels (FIGS. 2 and 3).
The B. ammoniagenes ppt1 gene was PCR amplified from isolated genomic DNA using the following primers: [0206]

16117 (forward):

5′-GTCGACATGCTCGACAACCGTGAAGCG-3′ [SEQ ID NO: 7]

and

16118 (reverse):

5′-AGATCTTCACTGGTGGCTTGCCGTAGATCG [SEQ ID NO: 8]

C-3′
The PCR-amplified fragment was then cloned into the commercially available cloning vector pCR-Blunt II TOPO (Invitrogen Corporation, Carlsbad, Calif.). The complete double stranded sequence of the full-length ppt1 gene (SEQ ID NO: 3) was determined using a Perkin Elmer ABI 377 DNA sequencer. The corresponding protein sequence (SEQ ID NO: 4) was predicted based on standard genetic code using the program Omiga and compared with the published ppt1 sequence. Alignment of both the nucleic acid and predicted amino acid sequences to the published sequences (Stuible et al., [0207] J. Bacteriol., 178:4787, 1996) revealed that the cloned ppt1 gene was identical to the published sequence.

EXAMPLE 2

This example describes the transformation of [0208] E. coli with fasA gene constructs for functional testing.
The full-length, sequence-confirmed [0209] B. ammoniagenes ppt1 gene in the pCR-Blunt II TOPO vector described in Example 1 was cut out of the pCR-Blunt II TOPO backbone as a Sal I/Bgl II fragment, and ligated into the SalI/BamHI sites, respectively, of pSU19 (Bartolome et al., Gene, 102(1):75-78, 1991). The SalI sites of both the ppt1 and the pSU19 fragments were blunt-ended with the Klenow fragment of DNA polymerase I prior to ligation to enable inframe insertion of the ppt1 coding sequence into the lacZ coding sequence of pSU19. The PPT1 protein was thus expressed in E. coli as a lacZ fusion protein upon induction of the lacZ promoter in pSU19 by the use of isopropyl-1-thio-β-D-galactopyranoside (IPTG). This ppt1-containing vector was then transformed into E. coli strain VCS257 from Stratagene (cat#200256-51), along with the mfFAS cosmid clone, described in Example 1, for functional testing.
Because the plasmid pSU19 has a pACYC184 origin of replication and conveys chloramphenicol resistance, the ppt1 expressing plasmid could be stably maintained along with the cosmid (ampicillin resistance) expressing the fasA gene. [0210]
Based on the published report of Stuible et al., [0211] Eur. J. Biochem., 248:481-487 (1997), the endogenous fasA promoter was used to express the mfFAS polypeptide encoded by fasA in E. coli. As a result, E. coli transformants containing the fasA cosmid alone, the pSU19/ppt1 construct alone, and both the fasA cosmid and the pSU19/ppt1 construct were made for functional testing. The full-length fasA gene was also subcloned as a PciI fragment from pMON70058 into the E. coli expression vector pQE60 (QIAGEN, Inc., Valencia, Calif.) to enable inducible expression from an E. coli promoter (pMON70081).

EXAMPLE 3

This example sets forth the functional testing of transgene activity in [0212] E. coli using enzymatic assays.
In order to assay the [0213] E. coli strains containing the fasA cosmid and ppt1 gene construct the fasA gene product was partially purified essentially as outlined in Kawaguchi et al., Methods in Enzymology, 71:120-127 (1981). Frozen cells from the strain containing either the fasA cosmid alone, the pSU19/ppt1 construct alone, both the fasA cosmid and the pSU19/ppt1 construct, or the untransformed cell line alone were thawed in 0.1M potassium phosphate buffer (˜1 ml/1 gm) and cells lysed by high speed mixing with glass beads. The supernatant was centrifuged at 105,000×g for 60 minutes and removed. Ammonium sulphate was slowly added to the supernatant to give a final concentration of 30% w/v followed by 30 minutes of stirring. A second centrifugation step (25,000×g) was performed and the precipitate was re-suspended in 0.5M potassium phosphate buffer before passing through a Sephadex G-25 column.
The fasA activity in each of the extracts was determined by a radiochemical assay at 37° C. for 15 minutes using the conditions outlined in Kawaguchi et al., (1981). The results of these assays (shown in FIG. 4) demonstrated that only when the fasA cosmid (FA) and the pSU19/ppt1 (P) construct were both present was there any measurable fasA activity. Furthermore, they demonstrated that the fasA gene that was cloned and used for preparation of Brassica transformation constructs did encode a functional fasA enzyme. [0214]

EXAMPLE 4

This example describes the construction of a plant binary vector for seed-specific expression of the fasA gene in canola plants. The construction is shown graphically in FIGS. 5 and 6. The vector pMON75201 was designed to produce seed-specific expression of the [0215] B. ammoniagenes fasA and ppt1 genes in canola.
The full-length, sequence-confirmed [0216] B. ammoniagenes ppt1 gene in the pCR-Blunt II TOPO vector described in Example 1 was cut out of the pCR-Blunt II TOPO backbone using the Sal I and Bgl II sites engineered into the PCR primers 16117 and 16118 (SEQ ID NOs: 7 and 8, respectively) used in the cloning and ligated to the Sal I and BamHI sites between the napin promoter (base pairs 407-2151 of the Brassica campestris napin gene, N5, GenBank Accession Number M64632) and the napin 3′ untranslated region (UTR), N3, (base pairs 2728-3982 of the Brassica campestris napin gene, GenBank Accession Number M64632) found in the plant/E. coli binary vector pCGN7770 (FIG. 5). The napin promoter/B. ammoniagenes ppt1/napin 3′ UTR cassette was combined with the B. ammoniagenes fasA gene for simultaneous expression in Brassica plants as described below.
The full-length, sequence-confirmed [0217] B. ammoniagenes fasA gene was removed from pMON70058 (described in Example 1 and FIG. 1) using the restriction enzymes NotI and SmaI and was ligated into the NotI and blunted Sse83871 restriction sites between a napin promoter and napin 3′ UTR (as described above) contained in a two T-DNA binary vector pMON67164. The Sse83871 site was blunt-ended by the action of Klenow fragment of DNA polymerase I. The resultant vector, containing the pMON67164 backbone and the B. ammoniagenes fasA gene flanked by the napin expression sequences, was digested with PacI, blunt-ended by the action of Klenow fragment of DNA polymerase I, and then digested with AscI. The AscI/PvuII fragment containing the napin promoter/B. ammoniagenes ppt1 gene/napin 3′ UTR cassette in pCGN7770 (described above) was then inserted into the PacI blunt/AscI sites to form pMON75201. pMON75201 is a two T-DNA vector containing both the B. ammoniagenes fasA gene and the B. ammoniagenes ppt1 gene each under the control of seed-specific napin expression sequences (napin promoter and 3′ UTR) and located within one set of T-DNA left and right borders. A selectable marker for plant transformation, containing the FMV ³⁵S promoter, (F35S, base pairs 6927-6474 of the FMV promoter which is the promoter for ORF VII, GenBank Accession Number X06166) driving a CP4 selectable marker gene (a chloroplast targeting sequence from the Arabidopsis EPSP gene linked to a synthetic EPSP synthase coding region as described in U.S. Pat. No. 5,633,435) and a E93′ UTR (Coruzzi et al., EMBO J., 3(8):1671-1679, 1984) is located within a second set of T-DNA left and right borders.

EXAMPLE 5

This example describes the transformation of canola plants with fasA and ppt1 genes. [0218]
Canola plants ([0219] Brassica napus) are transformed using a modification of the protocol described by Radke et al., Plant Cell Reports, 11:499-505 (1992). Briefly canola seed of the cultivar ‘Ebony’ (Monsanto Canada, Inc., Winnipeg, Canada) are disinfected and germinated in vitro as described in Radke et al., 1992. Precocultivation with tobacco feeder plates, explant preparation and inoculation of explants with Agrobacterium tumefaciens strain ABI (Koncz and Schell, Mol Gen Genet., 204:383-396, 1986) containing the vector pMON75201 are as described with the Agrobacterium being maintained in LB media (solid or liquid) containing 75 mg/l spectinomycin, 25 mg/l chloramphenicol, and 50 mg/l kanamycin. For plant transformation including callus induction, shoot regeneration, maturation and rooting, glyphosate selection is used rather than the kanamycin selection as described in Radke et al., 1992. Specifically, the B5-1 callus induction medium is supplemented with 500 mg/l carbenicillin and 50 mg/l Timentin (Duchefa Biochemie BV) to inhibit the Agrobacterium growth and kanamycin is omitted from the media. B5BZ shoot regeneration medium contains 500 mg/l carbenicillin, 50 mg/l Timentin, and 45 mg/l glyphosate with explants being transferred to fresh medium every 2 weeks. Glyphosate selected shoots are transferred to hormone-free B5-0 shoot maturation medium containing 300 mg/l carbenicillin and 45 mg/l glyphosate for 2 weeks and finally shoots are transferred to B5 root induction medium containing 45 mg/l glyphosate. Rooted green plantlets are transplanted to potting soil and acclimated to green house conditions. Plants are maintained in a greenhouse under standard conditions.
Developing seed is harvested at various stages after pollination and stored at −70° C. Mature seed is collected and stored under controlled conditions consisting of about 17° C. and 30% humidity. [0220]

EXAMPLE 6

This example describes the evaluation and selection of R2 seed from canola plants transformed with the fasA and ppt1 genes as described above in Example 5. [0221]
From the transformation of canola ex-plants with pMON75201, as described above, 110 events were generated. These events were analyzed for the presence of the gene of interest (GOI) by PCR, for transcription expression of the GOI by TaqMan methodology, and for the presence of the FasA protein by western blot analysis. The events testing positive for the GOI by PCR were considered for selection to advance in the development of high oil varieties. [0222]
The western blot analysis for the presence of the FasA protein was done using methods well known in the art. Briefly, antibodies were generated by a contract laboratory (Zymed Laboratories Inc., South San Francisco, Calif.) to 4 synthetic peptides located in different regions of the fasA gene; fasA[0223] _2843-2858=(C)SKHDTSTNANDPNESE (SEQ ID NO: 44),fasA_1755-1768=(C)QNKIRQDQINDSDT (SEQ ID NO: 45), fasA_915-930=(C)RINSDSYWDNLPEEQR (SEQ ID NO: 46), and fasA_1431-1444=(C)TLVERDENGNSNYG (SEQ ID NO: 47). A protein extract from each of the events was separated using SDS-PAGE according to Laemmli, Nature, 227: 680 (1970), and transferred to a polyvinylidene difluoride (PVDF) membrane in Tris buffered saline (TBS; 25 mM Tris, 150 mM NaCl) (BioRad, Bulletin #9016). The membrane was blocked with TBST (TBS with 0.05% Tween 20) containing 1% bovine serum albumin (BSA) for 10 minutes then incubated overnight at room temperature with a combined solution of primary antibodies from fasA_2843-2858, faSA_1755-1768, and fasA_1431-1444, at a 1:2000 dilution of each antibody in TBST with 1% BSA. The membrane was washed 3×15 minutes with TBST then exposed to a reporting, secondary antibody (Anti-rabbit-AP conjugate, Promega S3731, Madison, Wis., 1:5000 in TBST) for one hour. The membrane was washed 3×15 minutes with TBST followed by 2 minutes with TBS to remove residual Tween 20. Western Blue Stabilized Substrate for Alkaline Phosphatase (Promega S3841, Madison, Wis.) was added to visualize protein. Development was stopped by rinsing the membrane with purified water. Of the events tested, 23 were determined to be potentially western positive by exhibiting at least a weak response, and 8 of those confirmed to be positive by exhibiting a strong response. The results are shown in FIG. 7.
Mature R1 seed from all 23 events were planted and a selection test was performed to identify gene positive and gene negative lines for each event. For the event selection test, plants from each event were analyzed for the presence of the gene of interest (GOI) by PCR using primers from the GOI and promoter region. DNA for the PCR was isolated from leaf tissue using DNeasy 96 Plant Kit from Qiagen (Cat. No. 69181). The forward primer, located in the promoter region, was primer # 6456 (5′-TTCATAAGATGTCACGCCAGG-3′) (SEQ ID NO: 48), and the reverse primer, located in the GOI, was primer # 14873 (5′-GGTACGCGTCATATTCCTTG-3′) (SEQ ID NO: 49). PCR protocol was set at 97° C. for 1 minute, 40 cycles of [94° C. 15 seconds, 60° C. 30 seconds, 72° C. 30 seconds], and 72° C. for 5 minutes. Fourteen events (indicated by a in FIG. 7) were advanced to the R2 seed stage by growing R1 plants to maturity in the greenhouse under standard conditions. Ten of these events were identified as single locus (indicated by bin FIG. 7) and were also planted in field trials (Brawley, Calif.) as R1 transplants. The R2 seed from gene positive and null segregants from 4 events (BN_G1193, BN_G1198, BN_G1214, and BN_G1220) as well as commercial control lines, were planted in a randomized complete bloc experiment in field trials (Thief River Falls, Minn.). [0224]
In addition to the PCR gene confirmation and the western blot analysis, developing R1 seed (approximately 20/event) of the 23 events were analyzed for transcript expression of the genes of interest, fasA and ppt1, by TaqMan. TaqMan analysis was performed using the TaqMan One-Step RT-PCR Master Mix Reagents Kit and Protocol (#4310299 rev. C, Applied Biosystems, Foster City, Calif.). Low, medium, and high GOI-expressing lines are carried forward. Two separate experiments were performed on the same, pooled sample from each event. The results of both experiments are shown in tabular form in FIG. 7. [0225]
On the occasion when multiple gene copies of the GOI or marker are present, additional work is required to generate a null segregant from which phenotypic comparisons can be made. In addition to the original 14 events that were advanced, two GOI multicopy events (BN_G1223 and BN_G1239) were identified that exhibited a strong response in the western blot analysis. These events were crossed to the variety Ebony in order to produce gene positive as well as null segregants from an F1 population. Event BN_G1216 contained multiple copies of the marker gene, therefore it was out-crossed to Ebony as well. F1 transplants were generated for these events and were planted in the field and greenhouse. These 3 events are identified by [0226] ^cin FIG. 7. A comparison of the gene positive and null segregant selections is made in the F2 seed generation.
Oil and protein content of the F1 and R2 seed were established by near-infrared reflectance (NIR) spectroscopy (Williams and Norris, eds., Near-infrared Technology in the Agricultural and Food Industries, American Association of Cereal Chemists, Inc., St. Paul, Minn. (1987)), using a standard curve generated from analysis of canola seed with varying oil and protein levels. Briefly, mature canola seeds previously dried to less than 10% moisture were equilibrated to ambient humidity in paper envelopes at room temperature. Single replicate sub-samples (2-3 g) were placed in NIR ring cups (aluminum/quartz; 2 inch diameter by 0.5 inch thick, Foss North America Inc., Silver Springs, Md.) and sealed with a paperboard disk. The loaded ring cups were placed in an autoloader and scanned sequentially on a [0227] Foss Analytical model 6500 Spectrometer, (Foss North America Inc., Silver Springs, Md.). Each sample was scanned 25 times from 400 to 2500 nm (resolution 2 nm) and the average spectrum was compiled. The averaged spectrum was reduced to second derivative spectra, smoothed, and transformed to a series of principal component scores. The total oil and protein levels were predicted based on a previously prepared calibration models. Commercially available software (WinISI ver 1.00, Infrasoft International LLC, State College, Pa.) was used for calibration development and instrument operation.
One-way analysis of variance and the Student's T-test (JMP software, version 4.04, SAS Institute Inc., Cary, N.C.) was performed to identify significant differences between transgenic and non-transgenic seed pools as determined by transgene-specific PCR. As a result of the statistical analysis of the oil results, R2 seed from event BN_G1216 was determined to have a statistically significant increase in oil content as compared to a negative isoline control. The mean oil content determined by a one-way analysis of variance (ANOVA) for the positive isoline was 44.0%, as compared to 41.8% for the negative isoline control. The results are shown in FIG. 8. [0228]

EXAMPLE 7

This example describes the generation and evaluation of R3 seed and F2 seed from canola plants transformed with the fasA and ppt1 genes as described above in Example 5. [0229]
The event that showed a significant increase in oil in the greenhouse (BN_G1216) is included in a randomized complete bloc field test. This event did not show a positive phenotype as an R1 transplant in a first field trial (described above in Example 6). However, because field conditions are variable and have been shown to affect phenotype, and because of the positive phenotype in the greenhouse trial, this second field trial is determined to be warranted. In this second field trial, the gene positive line is compared to its null segregant to determine phenotype. Ebony is included in this experiment as a varietal control. The resulting seed is analyzed for oil and protein and the data is analyzed for statistical differences as described above in Example 6. The results of the R3 seed corroborate the results from the greenhouse grown R2 seed, described above in Example 6. [0230]
Three additional events, BN_G1223, BN_G1216, and BN_G1239, that were Western positive but contained multiple gene copies, are crossed to the variety Ebony in order to produce null segregants from an F1 population of seed that contained gene positive as well as the null segregants. The 3 out-crossed events, BN_G1233xEbony, BN_Gl239xEbony, and BN_Gl216xEbony, are grown in the field and greenhouse as F1 single plants. They are randomized as positive and negative selections and are grown with Ebony control lines. These events are individually isolated to produce selfed seed. The resulting F2 seed is harvested and analyzed for oil and protein, and the data is analyzed for statistical differences, as described above. The results of the F2 seed corroborate the results from the greenhouse grown R2 seed, described above in Example 6. [0231]
The present invention is not limited to the precise details shown and set forth hereinabove, for it should be understood that many variations and modifications may be made while still remaining within the spirit and scope of the present invention defined by the claims. [0232]
1 49 1 9122 DNA Brevibacterium ammoniagenes 1 atgtcgttga cccccttgca taccttgtct aatgacagca ctgctcccgc ggtgctgttt 60 gcgggtcagg gttctgcatg gcaaaaggcc atcgctgatg ccgcagccag ccctcaccag 120 ggcgcacatt gcgcgacatc ctaaaagaag ttcgcacgac caccggccca gtagcacgca 180 tcattgcgtc gtcgtgccct ggcgtttatg aacgcttgga agaacttgct cagacccccg 240 ctgaccaagc accgtggcca aggaatatga cgcgtacccg gcttactcca tccccggcat 300 cgtcctggga caaattggtg ccattgagca cctcgcgcag ctgggcatcg atgtcgattc 360 cgcgcagtta gcaggccact ccagggttca ttaggtgttg cagccgttaa ggatgcacgc 420 caggccctgg ctattgctgt tttgatgggt actgcagcag cggtgaccca gggcgcgaat 480 gattcccgca cccacatgct gtccgtgctg gcgtaccacg tgagatggtc gaagaatacc 540 tcgctggtga cgctgcgatt gccgtggtca acggccgcgt gcactttgca ctgtcgggta 600 ccccagagga tctggctaag accgagtcca acctccccag gctgccgagt cctacaacga 660 cgcgctggaa gaacgccgca tcggcggctc cgaaattaac ccagtcttcg acgtattggc 720 cgtggcactt cctttccacc acgcatcact gcaggatgca gcgatctgac cgtggactac 780 gccacccagt gtggcctgga cgctgagctt gcacgcgagc tggcagattc catcctggtt 840 cagccacata gctgggttga gaccgtggcc ggtctcaact ccacctaccg ctctccttag 900 accgtggtct gtcttcgttg actacacctt tgattgccgg caccggcaag gttgtggttc 960 cagctgctac gccagcggag cgcgataacc tggctacccc aggcactgag ctgcctccgc 1020 ggtgaactac gagaagttct caccaaagct catctccttg cccaacggca agtcctacac 1080 tcagactcgt ttctccgagt ggaccggcat gtcccccatc attttgggcg gcatgacgcc 1140 gacacgatgg atccgggcat cgttgccgca gcggccaacg gtggctactg gtcagagatg 1200 gccggtggcg gtcagtactc cgatgaagct tttaccatca acaaagacgg catgatggag 1260 ctgctggagc aggtcgcacc gcagcattta acaccatgtt ctttgaccgc tacctgtgga 1320 acctacagtt cggtgtcacc cgcatttgtt ccaaggcacg cgctaatggt gctgcgttta 1380 ccggcgtgac catctgcctg gtatcccaga gctggatgaa gccaaggaat tgctggacca 1440 gctcacctcc gatggctttc catacatctc tttcaagccg ggcaccacca agcagattca 1500 agactgcgtc gctatcgcag cggaaacccc acccaccgcg tcatcatcca aattgaagac 1560 gcccacgctg gtggccacca ctcctgggtg gatctggatg aaatgctgct ggctacctac 1620 gcatgtgccc gtgagcacga caacctggcc acactgttgg tggcggcatc cactccccag 1680 accgcgcatc ggaatacctg accggtacct ggtccaccaa gtacggtttg cccatcatgc 1740 cggttgatgg tgtcttcttg ggcaccgtag ccatggcgcc aaggaagcaa cggctaatga 1800 tgacgttaag cagttgctag ttgatacccc aggtatttcc ccagagacca atggcggttg 1860 ggtaggccga ctagatgccg acggcggcgt gtcctcctcc cagtccacct gttggctgac 1920 ttgcacgaga ttgataactc gtttgccaag gcctcgcgca tgatcacctc gatcccgatc 1980 gaggagtatg acgagcgtcg cgacgagatc attgctgctc tggacaagac ctcaagccat 2040 acttcggtga cctgtcggag atgacctacg aggattgggt cgctcgtttc gcagagcgcg 2100 cctacccttg ggtggatcca acctggcacg atcgtttcca cgatctgctc cagcgcgtaa 2160 agcgcgtctc aatgacgctg accacggcga catcgagacc ctattcccca cactcgacga 2220 ctccgagaac gcaccagagg cagtagccaa gctgctggct gcctacccga atgcaaagac 2280 caccaagtca acacccgcga tgaggcatgg ttccctaccc ttatccgcaa gcacgtcaag 2340 ccaatgccgt ggaccaccgc tattgacggt gacctgaagg aatggtttgc caaggacacc 2400 ctgtggcagg cccggaccca cgctacgacg cagacggcgt acgcatcatt ccaggaccgg 2460 tttcggttgc tggtatcacc aagaagaatg agcccgtcgc aaacctgctc ggtcgcttcg 2520 aagacgccac caccgcagcg ttaacgatgc cggcgtggca ccagttgagc tctactcccg 2580 cttggcttct gccaagaatg cagaagagtt cctgcgcaat gcaccaacca tcatgtggca 2640 cggtcacctc attgccaacc cggcgtatga gctgccagaa gaagcttttg acatcgtcga 2700 tgacggcgaa ggctttgcta ttcgcatcaa ctcgactcct acagggataa cctcccagaa 2760 gagcagcgtc cgttctacgt caagcacgtt gatatccccg ttgcgctgtc ggaagccgta 2820 gcaaccggtg cctcccctgt tgttgatgac gcgcgtttgc aaaggcagtc ttcgacctgc 2880 tcgcaggcgt tgctggtgtc gggtctatct ctgagaccgg cgataagatc accgaactgc 2940 cgaaggtcat cgaaggctct gtctccgaag aaaaccctta cggcctgtgg aatactcctt 3000 taccttgcct tctaccctgc tgaccgcaca caccgcggta accggcgctg ccttgggcac 3060 cgccaacgca ggcaccccag atgcgctggt tggcccctgc tggccagcaa tttaaccgcg 3120 ctgggcaccg gtcgattgac cgaagaacac ggtgagccag ccggcaccga cttcccggtc 3180 attgaaggcc tgctcaacgc agtccacctc gaccacgtcg tcgatgtgcg tgttcctctt 3240 ccgaactcgc aaagggtgaa aagggcgaag gcggtcgcat tgacgtcacc tcccgctgtg 3300 catccatcgc ggaatccaac tccggtcgca ttgtcaccgt ggaacttgag ttgtgggatg 3360 ccgcaactaa gaagttgtgg cgacgcagat gcagcgcttt gccatccgtg gccgcgctac 3420 cggcacctcc gttccggttt ctgcaccatc ctggggcggc ggcaagtctc aggacaagat 3480 tgagaccacc ccacgtcctt cgtggatcgc gccattgtca ccgcgccatc ggatatgacc 3540 ccattcgcgc tggtctccgg tgactacaac ccaattcaca cctccaccaa cgccgcgcgc 3600 ttggtcaacc tcgacgcccc acggtgcacg gcatgtggct atctgccacc gcgcagcacc 3660 tagctggcaa ccacggcacc gtggtgggtt ggacctattc catgtacggc atggtccagc 3720 tcaacgatga agtagaaatc accgtcgaag cgtaggccgc aagggcattc acgcagcatt 3780 cgaggtcacc tgccgcatcg acggcgaagt agtctcccgc ggccaggcgc tcatggcaca 3840 gccacgcacc gcttatgtct acccaggcca gggcatcagg ccgagggcat gggccgtggt 3900 gaccgcgatg cttcggcagc agcgcgtgag gtatggcgtc gtgcagaccg ccacacccgc 3960 accgcactgg gcttttctat tcgccagatc atcgatgaca acccaccgag ctcgtcgttc 4020 gcggcaccaa gttcgtccac cccaatggcg tgctgcactt aacgcagttc actcaggttg 4080 ccctcgcagt cgttgcttat gcacaaaccg agcgcctgcg cgaagcagat ctctgggcac 4140 caactccatg tacgccggtc actcactggg tgagtacacc gcgctggcat cgttggcgaa 4200 tatctttgac ctcgaagcgg ttatcgacat cgtctactcc cgtggctctg ccatgggacc 4260 ttggtcgaac gtgatgaaaa cggtaactcc aactacggca tgggcgcgct gcgtccaaac 4320 atgattggtg ttcccgcaga ccaggttgag gcctacatcg cgcagaccgc ggaagaaact 4380 ggcgattcct cgaaatcgtc aactacaaca tcgctggtca gcagtactcc atcgcgggta 4440 ccaaggctgg tttggccgcc ctgaagaaaa aggccaactc cgtcaaggac cgtgcttatg 4500 tcacggttcc agcatcgatg tacctttcca ctcccaggta ctgcgcgacg gcgttcctgc 4560 tttcgcagaa aagctcgatg aactgttgcc agaaaccttg gacctggacg ccctggtcgg 4620 ccgctacgtg ccgaacctgt ggcgctgcca ttcgagctga cccaggaatt tgtcgataag 4680 gtcaagcctt tggctccttc cggcaagctg gataacctca aggtcgaaga caccgatgag 4740 caagcccctt ctcgcctgct catgatgagc tattgtcctg gcagttcgca tcacctgtgc 4800 gctggattga aacccagcag ctgctctttg aagaagtaga ccagatcatc gaagtcggtc 4860 tcgcttcatc cccaacgctg accaacttgg ccagcgctcc atggatatcg ccggcgtgga 4920 cctcccggtc ttcaacgtcg aacgcgacca agaccaggtc atgctccaag acgttcagga 4980 agcaccagct gcctccttcg acgtcgagga aggagaggca cctcttcgac cgcagcgtct 5040 gaaaccccag gtgaatccgc tgcggcggcc tcggataata cccaggccat cccatcggct 5100 gagccacaaa cggtggcaga ggcaccagca ccatccgccg caccagtggc ggcacccgtg 5160 ccgcagatgc tcctgacctg ccatttaccg cagcagaagc catcatggtt ctgttcgctt 5220 tccagaacaa gatccgccag gaccagatca atgactcgga tacggtcgaa gagtcaccaa 5280 cggtgtctcc tcccgccgta accaactgtt gatggatatg tccgcagaaa atcgcgtgcc 5340 cgccattgac ggtgcagccg atgctgacgt ggcaaccttg cgtgagcgcg tcaagactgc 5400 cgctccgggc tactcgccat tcggcaccgt cttgtctgag gctataccgc tcgtctgcgc 5460 cagctcactg gtgcagcagg cgtcaagccg gcctacattt cagagcgcgt gaccggaact 5520 tgggggctgc ctatgtcctg ggcagcccac gttgaggctg aaatcttgct cgctcccgtg 5580 aagaagactc agtgcgcggt ggctccttgt ccaccgttcc ttccgcggcg tcgtcgaagg 5640 ccgatgtcga tgcgcttgtc gatgccgcgg tccaggccgt agccgcagca cacggcaccc 5700 ggtatcccat ggtgctgcga gtggcgccgg cggcggtgga gtcgtcgact ccgcagcctt 5760 ggatgcttac gcagatatcg tcaccggtga aaacggtgtc ctcgctactg ctgctcgcca 5820 ggttctgctc agctgggctt ggtcgaggaa gcccctgaga cccctgagac cgataacacc 5880 ttgttcgcga ccgtcgaggc cgagctgggt tccggttggg aaaagaccgt taccccatcc 5940 tttgacgcca agccgcagtg cttttcgatg accgctgggc gtctgctcgc gaagatctcg 6000 cccgcgtggc actcggcgag atcgacttgc cagtcaagcg tttccaggga accggagaga 6060 ccatcgccaa gcaagcggaa ggtgggcgga gaacaccgct gcttccactg gtgcgcacgc 6120 gaaggcaacc gctgccgaga ccctgcatgc tattgctgcc gcagcgcgcg aagaactcga 6180 cggcgaattc gctggcgatg tcgcgttgtc accggtgcag ccccaggctc cattgctacc 6240 gctctcgtag aacgcctgct ggaaggcggc gcgaccgtca tcatgactgc gtcacgtgtc 6300 agccagtccc gtaaggaatt tgcacgcaag ctctcgctgc acacgcgatt cctggcgctg 6360 ccctgtgggt tgttcctgcg aacttgcgct cctaccgcga tgttgatgct ctcattgact 6420 ggattggtaa tgagcagcgt gcctctgtcg gcaacgaagt cagatcacca agccagcgtt 6480 gaccccaacc ttggccttcc cattcgcggc accttccgtg tccggttctg tggccgatgc 6540 cggcccacag gctgaaaacc agactcgcct gctgctgtgg tctgttgacg caccatcgct 6600 ggtctgtcca acctggcgca gcaaggcgtg gatacccgct gccacattgt gctgcctggt 6660 tctccgaacc gcggcatgtt cggtggcgac ggcgcttacg gcgaagtcaa ggcagcttgg 6720 acgctatttt ggccaagtgg tctgcagaag caggctggcc agaaggtgtt accttggcac 6780 aagccaagat tggctgggtc tctggtacct ccctgatggg cggcaacgac gttctgattc 6840 cgcagcggaa gccgctggca tccacgtgtg ggacccagaa gagatttctt cccagctcat 6900 ctccctagct tccgaagaat cccgcgcgaa ggcagccgag gctccactag agctggatct 6960 gaccggtggc tgggctcgtc caacatctcc atctccgagc tggctgccca ggcccgcgag 7020 gacgccgagg cacaagctgc ttccggtgat aatgcagacg cagctgcgga agctcctgca 7080 gccacgattc cagcacgcct aatacccgtt cagtagagct gcctgcagcg ctaccggaag 7140 gtgaagtggg cgacgtaacc acggatctgg atgacatggt cgtcatcgca ggtgtcggcg 7200 aagtctcctc gtggggttcg ggcgtacccg ctttgaggca gaatatggct tgcagcgcga 7260 tggcgctgtg gacctgaccg ccgctggtgt cttggaattg gcatggatga ccggactgat 7320 ttcctggtcc aatgacccac gtccagcctg tacgacgaag agggcaccga agtcgatgaa 7380 gcagatatct acgctcgctt ccgcgacgag gttgtagctc gctccggtat ccgtaccttg 7440 accgataagt acaacatggt tgaccagggc tccattgcct gacttctgtg ttcttggacc 7500 gcgatatcgt cttcaccgtt cctaccgaac aagaagcact cgatattgaa gaagccgacc 7560 catcgtttac caagctgcgc gaagtcgacg gcgagtggga agtccccgtt tgaagggtgc 7620 caccgcccgc gtgccacgca aggcaacgtt gactcgtacc gttgctggtc aaatgccgga 7680 tcacttcgat gctgccaagt ggggcattcc agaccacatg ctggatgcac tgaccgcatg 7740 gccgtgtgga acctggtgac cgcagtcgat gcctttaccc aggcgggctt tagcccggct 7800 gagttgctgc aggttattca cccagcgcag gttgctacca cccagggcac cggtatcgcg 7860 gcatggaatc cctgcacaag gtcttcgtga cccgtctgct cggtgaagac cgtccttccg 7920 acatcctgca ggaagcactg cctaacgtta ttgcagcgca caccatgcag tctttggtgg 7980 gcggcacggt tcgatgattc accctatcgg tgcttgtgcc accgctgcgg tgtccatcga 8040 agaaggcgtg gacaagattg ccctgggcaa ggccgacctg gtcgttgccg gtggtatcga 8100 tgacgtccaa gttgagtctt tgaccggctt cggcgacatg aacgccaccg ctgagaccaa 8160 aagatgaccg atcagggcat tgatgaccgc ttcatctccc gtgcgaatga ccgccgtcgt 8220 ggcggcttcc tcgaggcaga aggcggcggt accgtgcttc tggttcgcgg ttccctggct 8280 cgtgagaggg tctgccggtc tacgcggtcg ttgcgcacga ggcgtcctac ggtgcccaca 8340 cctccattcc tgctccaggt ttgggtgctt tgggcgctgg ccgtggccgg aagaactccc 8400 gcctggccaa gggctggctg gtttgggtct gactccaaat gacgtctcgg tactgtccaa 8460 gcacgacacc tcgaccaacg ccaatgaccc gaatgagtcg gaactgcact ccatcttgtg 8520 gcctgctatt ggccgcgatg tgaccagcca ctgtttgtga tttcgcagaa gtcactgact 8580 ggtcactcca aggctggtgc cgcgctgttc cagaccggcg gtttgattga cgtcttccgc 8640 acgggacgca ttccagctaa cctgtcgcgg attgtgtgga tccattgatt gagccaaagg 8700 ccacgaactt ggtctggcta cgctccccac tagatgtgga agcagccaac cgcccggtca 8760 aggccgcggc gctcacctcg ctcggcttcg gtcactcggt gcattgattg tctacgcgca 8820 cccaggtgtc ttcgaggctg ccgttgccca gcaggtttcg gccgaggctg ctgccgaatg 8880 gcgcgagaag gcaaatgccc gcctcgccgc cggtgcagca cgttcgaagc cggcatgatt 8940 ggcaaggaaa ccttgttcga ggtcatcgac ggccgccgcc tgcctgacgc agcgggcacc 9000 gttgagattg agaactacgg cccagtcgcc gccgacaagg ccgcagaatg cgctcttgct 9060 tgacgacgac atccgtctta ccgccgaagg cactttccct ccggcgaagt aggacaaagt 9120 ag 9122 2 3040 PRT Brevibacterium ammoniagenes 2 Met Ser Leu Thr Pro Leu His Thr Leu Ser Asn Asp Ser Thr Ala Pro 1 5 10 15 Ala Val Leu Phe Ala Gly Gln Gly Ser Ala Trp Gln Lys Ala Ile Ala 20 25 30 Asp Ala Ala Ala Ser Pro His Gln Gly Ala Gln Leu Arg Asp Ile Leu 35 40 45 Lys Glu Val Arg Thr Thr Thr Gly Pro Val Ala Arg Ile Ile Ala Ser 50 55 60 Ser Cys Pro Gly Val Tyr Glu Arg Leu Glu Glu Leu Ala Gln Thr Pro 65 70 75 80 Ala Asp Gln Ala Pro Val Ala Lys Glu Tyr Asp Ala Tyr Pro Ala Tyr 85 90 95 Ser Ile Pro Gly Ile Val Leu Gly Gln Ile Gly Ala Ile Glu His Leu 100 105 110 Ala Gln Leu Gly Ile Asp Val Asp Ser Ala Gln Leu Ala Gly His Gln 115 120 125 Gly Ser Leu Gly Val Ala Ala Val Lys Asp Ala Arg Gln Ala Leu Ala 130 135 140 Ile Ala Val Leu Met Gly Thr Ala Ala Ala Val Thr Gln Gly Ala Asn 145 150 155 160 Asp Ser Arg Thr His Met Leu Ser Val Arg Gly Val Pro Arg Glu Met 165 170 175 Val Glu Glu Tyr Leu Ala Gly Asp Ala Ala Ile Ala Val Val Asn Gly 180 185 190 Arg Val His Phe Ala Leu Ser Gly Thr Pro Glu Asp Leu Ala Lys Thr 195 200 205 Glu Ser Asn Leu Thr Gln Ala Ala Glu Ser Tyr Asn Asp Ala Leu Glu 210 215 220 Glu Arg Arg Ile Gly Gly Ser Glu Ile Asn Pro Val Phe Asp Val Leu 225 230 235 240 Ala Val Ala Leu Pro Phe His His Ala Ser Leu Gln Asp Ala Asp Leu 245 250 255 Thr Val Asp Tyr Ala Thr Gln Cys Gly Leu Asp Ala Glu Leu Ala Arg 260 265 270 Glu Leu Ala Asp Ser Ile Leu Val Gln Pro His Ser Trp Val Glu Thr 275 280 285 Val Ala Gly Leu Asn Ser Thr Tyr Leu Leu Ser Leu Asp Arg Gly Leu 290 295 300 Ser Ser Leu Thr Thr Pro Leu Ile Ala Gly Thr Gly Lys Val Val Val 305 310 315 320 Pro Ala Ala Thr Pro Ala Glu Arg Asp Asn Leu Ala Thr Pro Gly Thr 325 330 335 Glu Leu Pro Thr Ala Val Asn Tyr Glu Lys Phe Ser Pro Lys Leu Ile 340 345 350 Ser Leu Pro Asn Gly Lys Ser Tyr Thr Gln Thr Arg Phe Ser Glu Trp 355 360 365 Thr Gly Met Ser Pro Ile Ile Leu Gly Gly Met Thr Pro Thr Met Asp 370 375 380 Pro Gly Ile Val Ala Ala Ala Ala Asn Gly Gly Tyr Trp Ser Glu Met 385 390 395 400 Ala Gly Gly Gly Gln Tyr Ser Asp Glu Ala Phe Thr Ile Asn Lys Asp 405 410 415 Gly Met Met Glu Leu Leu Glu Pro Gly Arg Thr Ala Ala Phe Asn Thr 420 425 430 Met Phe Phe Asp Arg Tyr Leu Trp Asn Leu Gln Phe Gly Val Thr Arg 435 440 445 Ile Cys Ser Lys Ala Arg Ala Asn Gly Ala Ala Phe Thr Gly Val Thr 450 455 460 Ile Cys Ala Gly Ile Pro Glu Leu Asp Glu Ala Lys Glu Leu Leu Asp 465 470 475 480 Gln Leu Thr Ser Asp Gly Phe Pro Tyr Ile Ser Phe Lys Pro Gly Thr 485 490 495 Thr Lys Gln Ile Gln Asp Cys Val Ala Ile Ala Ala Asn Pro Thr His 500 505 510 Arg Val Ile Ile Gln Ile Glu Asp Ala His Ala Gly Gly His His Ser 515 520 525 Trp Val Asp Leu Asp Glu Met Leu Leu Ala Thr Tyr Ala Cys Ala Arg 530 535 540 Glu His Asp Asn Leu Ala Ile Thr Val Gly Gly Gly Ile His Ser Pro 545 550 555 560 Asp Arg Ala Ser Glu Tyr Leu Thr Gly Thr Trp Ser Thr Lys Tyr Gly 565 570 575 Leu Pro Ile Met Pro Val Asp Gly Val Phe Leu Gly Thr Val Ala Met 580 585 590 Ala Thr Lys Glu Ala Thr Ala Asn Asp Asp Val Lys Gln Leu Leu Val 595 600 605 Asp Thr Pro Gly Ile Ser Pro Glu Thr Asn Gly Gly Trp Val Gly Arg 610 615 620 Leu Asp Ala Asp Gly Gly Val Ser Ser Ser Gln His Leu Leu Ala Asp 625 630 635 640 Leu His Glu Ile Asp Asn Ser Phe Ala Lys Ala Ser Arg Met Ile Thr 645 650 655 Ser Ile Pro Ile Glu Glu Tyr Asp Glu Arg Arg Asp Glu Ile Ile Ala 660 665 670 Ala Leu Asp Lys Thr Ser Lys Pro Tyr Phe Gly Asp Leu Ser Glu Met 675 680 685 Thr Tyr Glu Asp Trp Val Ala Arg Phe Ala Glu Arg Ala Tyr Pro Trp 690 695 700 Val Asp Pro Thr Trp His Asp Arg Phe His Asp Leu Leu Gln Arg Val 705 710 715 720 Glu Ala Arg Leu Asn Asp Ala Asp His Gly Asp Ile Glu Thr Leu Phe 725 730 735 Pro Thr Leu Asp Asp Ser Glu Asn Ala Pro Glu Ala Val Ala Lys Leu 740 745 750 Leu Ala Ala Tyr Pro Asn Ala Lys Thr Thr Val Asn Thr Arg Asp Glu 755 760 765 Ala Trp Phe Pro Thr Leu Ile Arg Lys His Val Lys Pro Met Pro Trp 770 775 780 Thr Thr Ala Ile Asp Gly Asp Leu Lys Glu Trp Phe Ala Lys Asp Thr 785 790 795 800 Leu Trp Gln Ala Gln Asp Pro Arg Tyr Asp Ala Asp Gly Val Arg Ile 805 810 815 Ile Pro Gly Pro Val Ser Val Ala Gly Ile Thr Lys Lys Asn Glu Pro 820 825 830 Val Ala Asn Leu Leu Gly Arg Phe Glu Asp Ala Thr Thr Ala Ala Leu 835 840 845 Asn Asp Ala Gly Val Ala Pro Val Glu Leu Tyr Ser Arg Leu Ala Ser 850 855 860 Ala Lys Asn Ala Glu Glu Phe Leu Arg Asn Ala Pro Thr Ile Met Trp 865 870 875 880 His Gly His Leu Ile Ala Asn Pro Ala Glu Leu Pro Glu Glu Ala Phe 885 890 895 Asp Ile Val Asp Asp Gly Glu Gly Phe Ala Ile Arg Ile Asn Ser Asp 900 905 910 Ser Tyr Arg Asp Asn Leu Pro Glu Glu Gln Arg Pro Phe Tyr Val Lys 915 920 925 His Val Asp Ile Pro Val Ala Leu Ser Glu Ala Val Ala Thr Gly Ala 930 935 940 Ser Pro Val Val Asp Asp Ala Arg Leu Pro Lys Ala Val Phe Asp Leu 945 950 955 960 Leu Ala Gly Val Ala Gly Val Gly Ser Ile Ser Glu Thr Gly Asp Lys 965 970 975 Ile Thr Glu Leu Pro Lys Val Ile Glu Gly Ser Val Ser Glu Glu Asn 980 985 990 Pro Tyr Gly Leu Val Glu Tyr Ser Phe Thr Leu Pro Ser Thr Leu Leu 995 1000 1005 Thr Ala His Thr Ala Val Thr Gly Ala Leu Gly Thr Ala Asn Ala 1010 1015 1020 Gly Thr Pro Asp Ala Leu Val Gly Pro Cys Trp Pro Ala Ile Tyr 1025 1030 1035 Thr Ala Leu Gly Thr Gly Arg Leu Thr Glu Glu His Gly Glu Pro 1040 1045 1050 Ala Gly Thr Asp Phe Pro Val Ile Glu Gly Leu Leu Asn Ala Val 1055 1060 1065 His Leu Asp His Val Val Asp Val Arg Val Pro Leu His Glu Leu 1070 1075 1080 Ala Lys Gly Glu Lys Gly Glu Gly Gly Arg Ile Asp Val Thr Ser 1085 1090 1095 Arg Cys Ala Ser Ile Ala Glu Ser Asn Ser Gly Arg Ile Val Thr 1100 1105 1110 Val Glu Leu Glu Leu Trp Asp Ala Ala Thr Gln Glu Val Val Ala 1115 1120 1125 Thr Gln Met Gln Arg Phe Ala Ile Arg Gly Arg Ala Thr Gly Thr 1130 1135 1140 Val Pro Val Ser Ala Pro Ser Trp Gly Gly Gly Lys Ser Gln Asp 1145 1150 1155 Lys Ile Glu Thr Thr Pro Arg Ser Phe Val Asp Arg Ala Ile Val 1160 1165 1170 Thr Ala Pro Ser Asp Met Thr Pro Phe Ala Leu Val Ser Gly Asp 1175 1180 1185 Tyr Asn Pro Ile His Thr Ser Thr Asn Ala Ala Arg Leu Val Asn 1190 1195 1200 Leu Asp Ala Pro Leu Val His Gly Met Trp Leu Ser Ala Thr Ala 1205 1210 1215 Gln His Leu Ala Gly Asn His Gly Thr Val Val Gly Trp Thr Tyr 1220 1225 1230 Ser Met Tyr Gly Met Val Gln Leu Asn Asp Glu Val Glu Ile Thr 1235 1240 1245 Val Glu Arg Val Gly Arg Lys Gly Ile His Ala Ala Phe Glu Val 1250 1255 1260 Thr Cys Arg Ile Asp Gly Glu Val Ser Arg Gly Gln Ala Leu Met 1265 1270 1275 Ala Gln Pro Arg Thr Ala Tyr Val Tyr Pro Gly Gln Gly Ile Gln 1280 1285 1290 Ala Glu Gly Met Gly Arg Gly Asp Arg Asp Ala Ser Ala Ala Ala 1295 1300 1305 Arg Glu Val Trp Arg Arg Ala Asp Arg His Thr Arg Thr Ala Leu 1310 1315 1320 Gly Phe Ser Ile Arg Gln Ile Ile Asp Asp Asn Pro Thr Glu Leu 1325 1330 1335 Val Val Arg Gly Thr Lys Phe Val His Pro Asn Gly Val Leu His 1340 1345 1350 Leu Thr Gln Phe Thr Gln Val Ala Leu Ala Val Val Ala Tyr Ala 1355 1360 1365 Gln Thr Glu Arg Leu Arg Glu Ala Asp Ala Leu Gly Thr Asn Ser 1370 1375 1380 Met Tyr Ala Gly His Ser Leu Gly Glu Tyr Thr Ala Leu Ala Leu 1385 1390 1395 Ala Asn Ile Phe Asp Leu Glu Ala Val Ile Asp Ile Val Tyr Ser 1400 1405 1410 Arg Gly Ser Ala Met Gly Thr Leu Val Glu Arg Asp Glu Asn Gly 1415 1420 1425 Asn Ser Asn Tyr Gly Met Gly Ala Leu Arg Pro Asn Met Ile Gly 1430 1435 1440 Val Pro Ala Asp Gln Val Glu Ala Tyr Ile Ala Gln Thr Ala Glu 1445 1450 1455 Glu Thr Gly Glu Phe Leu Glu Ile Val Asn Tyr Asn Ile Ala Gly 1460 1465 1470 Gln Gln Tyr Ser Ile Ala Gly Thr Lys Ala Gly Leu Ala Ala Leu 1475 1480 1485 Lys Lys Lys Ala Asn Ser Val Lys Asp Arg Ala Tyr Val Thr Val 1490 1495 1500 Pro Gly Ile Asp Val Pro Phe His Ser Gln Val Leu Arg Asp Gly 1505 1510 1515 Val Pro Ala Phe Ala Glu Leu Asp Glu Leu Leu Pro Glu Thr Leu 1520 1525 1530 Asp Leu Asp Ala Leu Val Gly Arg Tyr Val Pro Asn Leu Val Ala 1535 1540 1545 Leu Pro Phe Glu Leu Thr Gln Glu Phe Val Asp Lys Val Lys Pro 1550 1555 1560 Leu Ala Pro Ser Gly Lys Leu Asp Asn Leu Lys Val Glu Asp Thr 1565 1570 1575 Asp Glu Gln Ala Pro Ser Arg Leu Leu Met Ile Glu Leu Leu Ser 1580 1585 1590 Trp Gln Phe Ala Ser Pro Val Arg Trp Ile Glu Thr Gln Gln Leu 1595 1600 1605 Leu Phe Glu Glu Val Asp Gln Ile Ile Glu Val Gly Leu Ala Ser 1610 1615 1620 Ser Pro Thr Leu Thr Asn Leu Ala Lys Arg Ser Met Asp Ile Ala 1625 1630 1635 Gly Val Asp Leu Pro Val Phe Asn Val Glu Arg Asp Gln Gln Val 1640 1645 1650 Met Leu Gln Asp Val Gln Glu Ala Pro Ala Ala Ser Phe Asp Val 1655 1660 1665 Glu Glu Gly Glu Ala Thr Ser Ser Thr Ala Ala Ser Glu Thr Pro 1670 1675 1680 Gly Glu Ser Ala Ala Ala Ala Ser Asp Asn Thr Gln Ala Ile Pro 1685 1690 1695 Ser Ala Glu Pro Gln Thr Val Ala Glu Ala Pro Ala Pro Ser Ala 1700 1705 1710 Ala Pro Ala Gly Gly Thr Arg Ala Ala Asp Ala Pro Asp Leu Pro 1715 1720 1725 Phe Thr Ala Ala Glu Ala Ile Met Val Leu Phe Ala Phe Gln Asn 1730 1735 1740 Lys Ile Arg Gln Asp Gln Ile Asn Asp Ser Asp Thr Val Glu Glu 1745 1750 1755 Leu Thr Asn Gly Val Ser Ser Arg Arg Asn Gln Leu Leu Met Asp 1760 1765 1770 Met Ser Ala Glu Asn Val Pro Ala Ile Asp Gly Ala Ala Asp Ala 1775 1780 1785 Asp Val Ala Thr Leu Arg Glu Arg Val Lys Thr Ala Ala Pro Gly 1790 1795 1800 Tyr Ser Pro Phe Gly Thr Val Leu Ser Glu Ala Ile Thr Ala Arg 1805 1810 1815 Leu Arg Gln Leu Thr Gly Ala Ala Gly Val Lys Pro Ala Tyr Ile 1820 1825 1830 Ser Glu Arg Val Thr Gly Thr Trp Gly Leu Pro Met Ser Trp Ala 1835 1840 1845 Ala His Val Glu Ala Glu Ile Leu Leu Gly Ser Arg Glu Glu Asp 1850 1855 1860 Ser Val Arg Gly Gly Ser Leu Ser Thr Val Pro Ser Ala Ala Ser 1865 1870 1875 Ser Lys Ala Asp Val Asp Ala Leu Val Asp Ala Ala Val Gln Ala 1880 1885 1890 Val Ala Ala Ala His Gly Thr Ser Val Ser His Gly Ala Ser Gly 1895 1900 1905 Ala Gly Gly Gly Gly Val Val Asp Ser Ala Ala Leu Asp Ala Tyr 1910 1915 1920 Ala Asp Ile Val Thr Gly Glu Asn Gly Val Leu Ala Thr Ala Ala 1925 1930 1935 Arg Gln Val Leu Ala Gln Leu Gly Leu Val Glu Glu Ala Pro Glu 1940 1945 1950 Thr Pro Glu Thr Asp Asn Thr Leu Phe Ala Thr Val Glu Ala Glu 1955 1960 1965 Leu Gly Ser Gly Trp Glu Lys Thr Val Thr Pro Ser Phe Asp Ala 1970 1975 1980 Lys Arg Ala Val Leu Phe Asp Asp Arg Trp Ala Ser Ala Arg Glu 1985 1990 1995 Asp Leu Ala Arg Val Ala Leu Gly Glu Ile Asp Leu Pro Val Lys 2000 2005 2010 Arg Phe Gln Gly Thr Gly Glu Thr Ile Ala Lys Gln Ala Glu Trp 2015 2020 2025 Trp Ala Glu Asn Ala Ala Ser Thr Gly Ala His Ala Lys Ala Thr 2030 2035 2040 Ala Ala Glu Thr Leu His Ala Ile Ala Ala Ala Ala Arg Glu Glu 2045 2050 2055 Leu Asp Gly Glu Phe Ala Gly Asp Val Ala Leu Val Thr Gly Ala 2060 2065 2070 Ala Pro Gly Ser Ile Ala Thr Ala Leu Val Glu Arg Leu Leu Glu 2075 2080 2085 Gly Gly Ala Thr Val Ile Met Thr Ala Ser Arg Val Ser Gln Ser 2090 2095 2100 Arg Lys Glu Phe Ala Arg Lys Leu Tyr Ala Ala His Ala Ile Pro 2105 2110 2115 Gly Ala Ala Leu Trp Val Val Pro Ala Asn Leu Arg Ser Tyr Arg 2120 2125 2130 Asp Val Asp Ala Leu Ile Asp Trp Ile Gly Asn Glu Gln Arg Ala 2135 2140 2145 Ser Val Gly Asn Glu Val Lys Ile Thr Lys Pro Leu Thr Pro Thr 2150 2155 2160 Leu Ala Phe Pro Phe Ala Ala Pro Ser Val Ser Gly Ser Val Ala 2165 2170 2175 Asp Ala Gly Pro Gln Ala Glu Asn Gln Thr Arg Leu Leu Leu Trp 2180 2185 2190 Ser Val Glu Arg Thr Ile Ala Gly Leu Ser Asn Leu Ala Gln Gln 2195 2200 2205 Gly Val Asp Thr Arg Cys His Ile Val Leu Pro Gly Ser Pro Asn 2210 2215 2220 Arg Gly Met Phe Gly Gly Asp Gly Ala Tyr Gly Glu Val Lys Ala 2225 2230 2235 Ala Leu Asp Ala Ile Leu Ala Lys Trp Ser Ala Glu Ala Gly Trp 2240 2245 2250 Pro Glu Gly Val Thr Leu Ala Gln Ala Lys Ile Gly Trp Val Ser 2255 2260 2265 Gly Thr Ser Leu Met Gly Gly Asn Asp Val Leu Ile Pro Ala Ala 2270 2275 2280 Glu Ala Ala Ile His Val Trp Asp Pro Glu Glu Ile Ser Ser Gln 2285 2290 2295 Leu Ile Ser Leu Ala Ser Glu Glu Ser Arg Ala Lys Ala Ala Glu 2300 2305 2310 Ala Pro Leu Glu Leu Asp Leu Thr Gly Gly Leu Gly Ser Ser Asn 2315 2320 2325 Ile Ser Ile Ser Glu Leu Ala Ala Gln Ala Arg Glu Asp Ala Glu 2330 2335 2340 Ala Gln Ala Ala Ser Gly Asp Asn Ala Asp Ala Ala Ala Glu Ala 2345 2350 2355 Pro Ala Ala Thr Ile Pro Ala Leu Pro Asn Thr Arg Ser Val Glu 2360 2365 2370 Leu Pro Ala Ala Leu Pro Glu Gly Glu Val Gly Asp Val Thr Thr 2375 2380 2385 Asp Leu Asp Asp Met Val Val Ile Ala Gly Val Gly Glu Val Ser 2390 2395 2400 Ser Trp Gly Ser Gly Arg Thr Arg Phe Glu Glu Tyr Gly Leu Gln 2405 2410 2415 Arg Asp Gly Ala Val Asp Leu Thr Ala Ala Gly Val Leu Glu Leu 2420 2425 2430 Ala Trp Met Thr Gly Leu Ile Ser Trp Ser Asn Asp Pro Arg Pro 2435 2440 2445 Ala Trp Tyr Asp Glu Glu Gly Thr Glu Val Asp Glu Ala Asp Ile 2450 2455 2460 Tyr Ala Arg Phe Arg Asp Glu Val Val Ala Arg Ser Gly Ile Arg 2465 2470 2475 Thr Leu Thr Asp Lys Tyr Asn Met Val Asp Gln Gly Ser Ile Asp 2480 2485 2490 Leu Thr Ser Val Phe Leu Asp Arg Asp Ile Val Phe Thr Val Pro 2495 2500 2505 Thr Glu Gln Glu Ala Leu Asp Ile Glu Glu Ala Asp Pro Ser Phe 2510 2515 2520 Thr Lys Leu Arg Glu Val Asp Gly Glu Trp Glu Val Thr Arg Leu 2525 2530 2535 Lys Gly Thr Ala Arg Val Pro Arg Lys Ala Thr Leu Thr Arg Thr 2540 2545 2550 Val Ala Gly Gln Met Pro Asp His Phe Asp Ala Ala Lys Trp Gly 2555 2560 2565 Ile Pro Asp His Met Leu Asp Ala Leu Asp Arg Met Ala Val Trp 2570 2575 2580 Asn Leu Val Thr Ala Val Asp Ala Phe Thr Gln Ala Gly Phe Ser 2585 2590 2595 Pro Ala Glu Leu Leu Gln Val Ile His Pro Ala Gln Val Ala Thr 2600 2605 2610 Thr Gln Gly Thr Gly Ile Gly Gly Met Glu Ser Leu His Lys Val 2615 2620 2625 Phe Val Thr Arg Leu Leu Gly Glu Asp Arg Pro Ser Asp Ile Leu 2630 2635 2640 Gln Glu Ala Leu Pro Asn Val Ile Ala Ala His Thr Met Gln Ser 2645 2650 2655 Leu Val Gly Gly Tyr Gly Ser Met Ile Pro Ile Gly Ala Cys Ala 2660 2665 2670 Thr Ala Ala Val Ser Ile Glu Glu Gly Val Asp Lys Ile Ala Leu 2675 2680 2685 Gly Lys Ala Asp Leu Val Val Ala Gly Gly Ile Asp Asp Val Gln 2690 2695 2700 Val Glu Ser Leu Thr Gly Phe Gly Asp Met Asn Ala Thr Ala Glu 2705 2710 2715 Thr Lys Lys Met Thr Asp Gln Gly Ile Asp Asp Arg Phe Ile Ser 2720 2725 2730 Arg Ala Asn Asp Arg Arg Arg Gly Gly Phe Leu Glu Ala Glu Gly 2735 2740 2745 Gly Gly Thr Val Leu Leu Val Arg Gly Ser Leu Ala Arg Glu Met 2750 2755 2760 Gly Leu Pro Val Tyr Ala Val Val Ala His Glu Ala Ser Tyr Gly 2765 2770 2775 Ala His Thr Ser Ile Pro Ala Pro Gly Leu Gly Ala Leu Gly Ala 2780 2785 2790 Gly Arg Gly Arg Lys Asn Ser Arg Leu Ala Lys Gly Leu Ala Gly 2795 2800 2805 Leu Gly Leu Thr Pro Asn Asp Val Ser Val Leu Ser Lys His Asp 2810 2815 2820 Thr Ser Thr Asn Asn Asp Pro Asn Glu Ser Glu Leu His Ser Ile 2825 2830 2835 Leu Trp Pro Ala Ile Gly Arg Asp Val Asp Gln Pro Leu Phe Val 2840 2845 2850 Ile Ser Gln Lys Ser Leu Thr Gly His Ser Lys Ala Gly Ala Ala 2855 2860 2865 Leu Phe Gln Thr Gly Gly Leu Ile Asp Val Phe Arg Thr Gly Arg 2870 2875 2880 Ile Pro Ala Asn Leu Ser Leu Asp Cys Val Asp Pro Leu Ile Glu 2885 2890 2895 Pro Lys Ala Thr Asn Leu Val Trp Leu Arg Ser Pro Leu Asp Val 2900 2905 2910 Glu Ala Ala Asn Arg Pro Val Lys Ala Ala Ala Leu Thr Ser Leu 2915 2920 2925 Gly Phe Gly His Val Gly Ala Leu Ile Val Tyr Ala His Pro Gly 2930 2935 2940 Val Phe Glu Ala Ala Val Ala Gln Gln Val Ser Glu Ala Ala Ala 2945 2950 2955 Glu Trp Arg Glu Lys Ala Asn Ala Arg Leu Ala Ala Gly Ala Ala 2960 2965 2970 Arg Phe Glu Ala Gly Met Ile Gly Lys Glu Thr Leu Phe Glu Val 2975 2980 2985 Ile Asp Gly Arg Arg Leu Pro Asp Ala Ala Gly Thr Val Glu Ile 2990 2995 3000 Glu Asn Tyr Gly Pro Val Ala Ala Asp Lys Ala Ala Glu Leu Arg 3005 3010 3015 Ser Cys Leu Thr Thr Thr Ser Val Leu Pro Pro Lys Ala Leu Ser 3020 3025 3030 Leu Arg Arg Ser Arg Thr Lys 3035 3040 3 459 DNA Brevibacterium ammoniagenes 3 gtgctcgaca accgtgaagc gatgaccgtg ggtgtggact tggtccacat ccccggcttt 60 gccgagcaat tgtcgcgccc tggttcgact tttgagcaag tgttttcgcc gttggaacgt 120 cgtcatgtca aacgcgccgt gacgctgcag cggatgctac gaattcgagc cttgcgggtt 180 cacggactga gcacctggct gggcggtggg cggcaaaaga agcgttcatc aaggcgtggt 240 cgcaagcgat ctacgcaagc caccagtgat tgaaccagac ctggtgaact tcgcagagat 300 cgaagtcttg cccgaccgct ggggcagggt agcgctgcag cttaaaggtg aagttgctgc 360 aaaacttcag gaatcaatag ggacgtggag ctggcgctga gcatcagcca tgatggcgat 420 tacgccaccg cgcagtgcct gctgcggtac cagcggtaa 459 4 152 PRT Brevibacterium ammoniagenes 4 Met Leu Asp Asn Arg Glu Ala Met Thr Val Gly Val Asp Leu Val His 1 5 10 15 Ile Pro Gly Phe Ala Glu Gln Leu Ser Arg Pro Gly Ser Thr Phe Glu 20 25 30 Gln Val Phe Ser Pro Leu Glu Arg Arg His Ala Gln Thr Arg Arg Asp 35 40 45 Ala Ala Ala Asp Ala Thr Asn Ser Ser Leu Ala Gly Ser Arg Thr Glu 50 55 60 His Leu Ala Gly Arg Trp Ala Ala Lys Glu Ala Phe Ile Lys Ala Trp 65 70 75 80 Ser Gln Ala Ile Tyr Gly Lys Pro Pro Val Ile Glu Pro Asp Leu Val 85 90 95 Asn Phe Ala Glu Ile Glu Val Leu Pro Asp Arg Trp Gly Arg Val Ala 100 105 110 Leu Gln Leu Lys Gly Glu Val Ala Ala Lys Leu Gln Glu Ser Ile Asp 115 120 125 Val Glu Leu Ala Leu Ser Ile Ser His Asp Gly Asp Tyr Ala Thr Ala 130 135 140 Gln Cys Leu Leu Arg Tyr Gln Arg 145 150 5 20 DNA Artificial PCR primer sequence 5 ccagctcaac gatgaagtag 20 6 20 DNA Artificial PCR primer sequence 6 tcgatgatct ggtctacttc 20 7 27 DNA Artificial Primer Sequence 7 gtcgacatgc tcgacaaccg tgaagcg 27 8 31 DNA Artificial Primer Sequence 8 agatcttcac tggtggcttg ccgtagatcg c 31 9 37 DNA Artificial Primer Sequence 9 tctagatgca tagttaacat gtcgttgacc cccttgc 37 10 20 DNA Artificial Primer Sequence 10 ggtacgcgtc atattccttg 20 11 40 DNA Artificial Primer Sequence 11 caaggaatat gacgcgtacc ctcgaggcag aaggcggcgg 40 12 37 DNA Artificial Primer Sequence 12 atgcatgtta acatgtctac tttgtcctac ttcgccg 37 13 33 DNA Artificial Primer Sequence 13 gtcgacatgc atatgctcga caaccgtgaa gcg 33 14 30 DNA Artificial Primer Sequence 14 agatctatgc attaccgctg gtaccgcagc 30 15 2073 PRT Schizosaccharomyces pombe 15 Met Val Glu Ala Glu Gln Val His Gln Ser Leu Arg Ser Leu Val Leu 1 5 10 15 Ser Tyr Ala His Phe Ser Pro Ser Ile Leu Ile Pro Ala Ser Gln Tyr 20 25 30 Leu Leu Ala Ala Gln Leu Arg Asp Glu Phe Leu Ser Leu His Pro Ala 35 40 45 Pro Ser Ala Glu Ser Val Glu Lys Glu Gly Ala Glu Leu Glu Phe Glu 50 55 60 His Glu Leu His Leu Leu Ala Gly Phe Leu Gly Leu Ile Ala Ala Lys 65 70 75 80 Glu Glu Glu Thr Pro Gly Gln Tyr Thr Gln Leu Leu Arg Ile Ile Thr 85 90 95 Leu Glu Phe Glu Arg Thr Phe Leu Ala Gly Asn Glu Val His Ala Val 100 105 110 Val His Ser Leu Gly Leu Asn Ile Pro Ala Gln Lys Asp Val Val Arg 115 120 125 Phe Tyr Tyr His Ser Cys Ala Leu Ile Gly Gln Thr Thr Lys Phe His 130 135 140 Gly Ser Ala Leu Leu Asp Glu Ser Ser Val Lys Leu Ala Ala Ile Phe 145 150 155 160 Gly Gly Gln Gly Tyr Glu Asp Tyr Phe Asp Glu Leu Ile Glu Leu Tyr 165 170 175 Glu Val Tyr Ala Pro Phe Ala Ala Glu Leu Ile Gln Val Leu Ser Lys 180 185 190 His Leu Phe Thr Leu Ser Gln Asn Glu Gln Ala Ser Lys Val Tyr Ser 195 200 205 Lys Gly Leu Asn Val Leu Asp Trp Leu Ala Gly Glu Arg Pro Glu Arg 210 215 220 Asp Tyr Leu Val Ser Ala Pro Val Ser Leu Pro Leu Val Gly Leu Thr 225 230 235 240 Gln Leu Val His Phe Ser Val Thr Ala Gln Ile Leu Gly Leu Asn Pro 245 250 255 Gly Glu Leu Ala Ser Arg Phe Ser Ala Ala Ser Gly His Ser Gln Gly 260 265 270 Ile Val Val Ala Ala Ala Val Ser Ala Ser Thr Asp Ser Ala Ser Phe 275 280 285 Met Glu Asn Ala Lys Val Ala Leu Thr Thr Leu Phe Trp Ile Gly Val 290 295 300 Arg Ser Gln Gln Thr Phe Pro Thr Thr Thr Leu Pro Pro Ser Val Val 305 310 315 320 Ala Asp Ser Leu Ala Ser Ser Glu Gly Asn Pro Thr Pro Met Leu Ala 325 330 335 Val Arg Asp Leu Pro Ile Glu Thr Leu Asn Lys His Ile Glu Thr Thr 340 345 350 Asn Thr His Leu Pro Glu Asp Arg Lys Val Ser Leu Ser Leu Val Asn 355 360 365 Gly Pro Arg Ser Phe Val Val Ser Gly Pro Ala Arg Ser Leu Tyr Gly 370 375 380 Leu Asn Leu Ser Leu Arg Lys Glu Lys Ala Asp Gly Gln Asn Gln Ser 385 390 395 400 Arg Ile Pro His Ser Lys Arg Lys Leu Arg Phe Ile Asn Arg Phe Leu 405 410 415 Ser Ile Ser Val Pro Phe His Ser Pro Tyr Leu Ala Pro Val Arg Ser 420 425 430 Leu Leu Glu Lys Asp Leu Gln Gly Leu Gln Phe Ser Ala Leu Lys Val 435 440 445 Pro Val Tyr Ser Thr Asp Asp Ala Gly Asp Leu Arg Phe Glu Gln Pro 450 455 460 Ser Lys Leu Leu Leu Ala Leu Ala Val Met Ile Thr Glu Lys Val Val 465 470 475 480 His Trp Glu Glu Ala Cys Gly Phe Pro Asp Val Thr His Ile Ile Asp 485 490 495 Phe Gly Pro Gly Gly Ile Ser Gly Val Gly Ser Leu Thr Arg Ala Asn 500 505 510 Lys Asp Gly Gln Gly Val Arg Val Ile Val Ala Asp Ser Phe Glu Ser 515 520 525 Leu Asp Met Gly Ala Lys Phe Glu Ile Phe Asp Arg Asp Ala Lys Ser 530 535 540 Ile Glu Phe Ala Pro Asn Trp Val Lys Leu Tyr Ser Pro Lys Leu Val 545 550 555 560 Lys Asn Lys Leu Gly Arg Val Tyr Val Asp Thr Arg Leu Ser Arg Met 565 570 575 Leu Gly Leu Pro Pro Leu Trp Val Ala Gly Met Thr Pro Thr Ser Val 580 585 590 Pro Trp Gln Phe Cys Ser Ala Ile Ala Lys Ala Gly Phe Thr Tyr Glu 595 600 605 Leu Ala Gly Gly Gly Tyr Phe Asp Pro Lys Met Met Arg Glu Ala Ile 610 615 620 His Lys Leu Ser Leu Asn Ile Pro Pro Gly Ala Gly Ile Cys Val Asn 625 630 635 640 Val Ile Tyr Ile Asn Pro Arg Thr Tyr Ala Trp Gln Ile Pro Leu Ile 645 650 655 Arg Asp Met Val Ala Glu Gly Tyr Pro Ile Arg Gly Val Thr Ile Ala 660 665 670 Ala Gly Ile Pro Ser Leu Glu Val Ala Asn Glu Leu Ile Ser Thr Leu 675 680 685 Gly Val Gln Tyr Leu Cys Leu Lys Pro Gly Ser Val Glu Ala Val Asn 690 695 700 Ala Val Ile Ser Ile Ala Lys Ala Asn Pro Thr Phe Pro Ile Val Leu 705 710 715 720 Gln Trp Thr Gly Gly Arg Ala Gly Gly His His Ser Phe Glu Asp Phe 725 730 735 His Ser Pro Ile Leu Leu Thr Tyr Ser Ala Ile Arg Arg Cys Asp Asn 740 745 750 Ile Val Leu Ile Ala Gly Ser Gly Phe Gly Gly Ala Asp Asp Thr Glu 755 760 765 Pro Tyr Leu Thr Gly Glu Trp Ser Ala Ala Phe Lys Leu Pro Pro Met 770 775 780 Pro Phe Asp Gly Ile Leu Phe Gly Ser Arg Leu Met Val Ala Lys Glu 785 790 795 800 Ala His Thr Ser Leu Ala Ala Lys Glu Ala Ile Val Ala Ala Lys Gly 805 810 815 Val Asp Asp Ser Glu Trp Glu Lys Thr Tyr Asp Gly Pro Thr Gly Gly 820 825 830 Ile Val Thr Val Leu Ser Glu Leu Gly Glu Pro Ile His Lys Leu Ala 835 840 845 Thr Arg Gly Ile Met Phe Trp Lys Glu Leu Asp Asp Thr Ile Phe Ser 850 855 860 Leu Pro Arg Pro Lys Arg Leu Pro Ala Leu Leu Ala Lys Lys Gln Tyr 865 870 875 880 Ile Ile Lys Arg Leu Asn Asp Asp Phe Gln Lys Val Tyr Phe Pro Ala 885 890 895 His Ile Val Glu Gln Val Ser Pro Glu Lys Phe Lys Phe Glu Ala Val 900 905 910 Asp Ser Val Glu Asp Met Thr Tyr Ala Glu Leu Leu Tyr Arg Ala Ile 915 920 925 Asp Leu Met Tyr Val Thr Lys Glu Lys Arg Trp Ile Asp Val Thr Leu 930 935 940 Arg Thr Phe Thr Gly Lys Leu Met Arg Arg Ile Glu Glu Arg Phe Thr 945 950 955 960 Gln Asp Val Gly Lys Thr Thr Leu Ile Glu Asn Phe Glu Asp Leu Asn 965 970 975 Asp Pro Tyr Pro Val Ala Ala Arg Phe Leu Asp Ala Tyr Pro Glu Ala 980 985 990 Ser Thr Gln Asp Leu Asn Thr Gln Asp Ala Gln Phe Phe Tyr Ser Leu 995 1000 1005 Cys Ser Asn Pro Phe Gln Lys Pro Val Pro Phe Ile Pro Ala Ile 1010 1015 1020 Asp Asp Thr Phe Glu Phe Tyr Phe Lys Lys Asp Ser Leu Trp Gln 1025 1030 1035 Ser Glu Asp Leu Ala Ala Val Val Gly Glu Asp Val Gly Arg Val 1040 1045 1050 Ala Ile Leu Gln Gly Pro Met Ala Ala Lys His Ser Thr Lys Val 1055 1060 1065 Asn Glu Pro Ala Lys Glu Leu Leu Asp Gly Ile Asn Glu Thr His 1070 1075 1080 Ile Gln His Phe Ile Lys Lys Phe Tyr Ala Gly Asp Glu Lys Lys 1085 1090 1095 Ile Pro Ile Val Glu Tyr Phe Gly Gly Val Pro Pro Val Asn Val 1100 1105 1110 Ser His Lys Ser Leu Glu Ser Val Ser Val Thr Glu Glu Ala Gly 1115 1120 1125 Ser Lys Val Tyr Lys Leu Pro Glu Ile Gly Ser Asn Ser Ala Leu 1130 1135 1140 Pro Ser Lys Lys Leu Trp Phe Glu Leu Leu Ala Gly Pro Glu Tyr 1145 1150 1155 Thr Trp Phe Arg Ala Ile Phe Thr Thr Gln Arg Val Ala Lys Gly 1160 1165 1170 Trp Lys Leu Glu His Asn Pro Val Arg Arg Ile Phe Ala Pro Arg 1175 1180 1185 Tyr Gly Gln Arg Ala Val Val Lys Gly Lys Asp Asn Asp Thr Val 1190 1195 1200 Val Glu Leu Tyr Glu Thr Gln Ser Gly Asn Tyr Val Leu Ala Ala 1205 1210 1215 Arg Leu Ser Tyr Asp Gly Glu Thr Ile Val Val Ser Met Phe Glu 1220 1225 1230 Asn Arg Asn Ala Leu Lys Lys Glu Val His Leu Asp Phe Leu Phe 1235 1240 1245 Lys Tyr Glu Pro Ser Ala Gly Tyr Ser Pro Val Ser Glu Ile Leu 1250 1255 1260 Asp Gly Arg Asn Asp Arg Ile Lys His Phe Tyr Trp Ala Leu Trp 1265 1270 1275 Phe Gly Glu Glu Pro Tyr Pro Glu Asn Ala Ser Ile Thr Asp Thr 1280 1285 1290 Phe Thr Gly Pro Glu Val Thr Val Thr Gly Asn Met Ile Glu Asp 1295 1300 1305 Phe Cys Arg Thr Val Gly Asn His Asn Glu Ala Tyr Thr Lys Arg 1310 1315 1320 Ala Ile Arg Lys Arg Met Ala Pro Met Asp Phe Ala Ile Val Val 1325 1330 1335 Gly Trp Gln Ala Ile Thr Lys Ala Ile Phe Pro Lys Ala Ile Asp 1340 1345 1350 Gly Asp Leu Leu Arg Leu Val His Leu Ser Asn Ser Phe Arg Met 1355 1360 1365 Val Gly Ser His Ser Leu Met Glu Gly Asp Lys Val Thr Thr Ser 1370 1375 1380 Ala Ser Ile Ile Ala Ile Leu Asn Asn Asp Ser Gly Lys Thr Val 1385 1390 1395 Thr Val Lys Gly Thr Val Tyr Arg Asp Gly Lys Glu Val Ile Glu 1400 1405 1410 Val Ile Ser Arg Phe Leu Tyr Arg Gly Thr Phe Thr Asp Phe Glu 1415 1420 1425 Asn Thr Phe Glu His Thr Gln Glu Thr Pro Met Gln Leu Thr Leu 1430 1435 1440 Ala Thr Pro Lys Asp Val Ala Val Leu Gln Ser Lys Ser Trp Phe 1445 1450 1455 Gln Leu Leu Asp Pro Ser Gln Asp Leu Ser Gly Ser Ile Leu Thr 1460 1465 1470 Phe Arg Leu Asn Ser Tyr Val Arg Phe Lys Asp Gln Lys Val Lys 1475 1480 1485 Ser Ser Val Glu Thr Lys Gly Ile Val Leu Ser Glu Leu Pro Ser 1490 1495 1500 Lys Ala Ile Ile Gln Val Ala Ser Val Asp Phe Gln Ser Val Asp 1505 1510 1515 Cys His Gly Asn Pro Val Ile Glu Phe Leu Lys Arg Asn Gly Lys 1520 1525 1530 Pro Ile Glu Gln Pro Val Glu Phe Glu Asn Gly Gly Tyr Ser Val 1535 1540 1545 Ile Gln Val Met Asp Glu Gly Tyr Ser Pro Val Phe Val Thr Pro 1550 1555 1560 Pro Thr Asn Ser Pro Tyr Ala Glu Val Ser Gly Asp Tyr Asn Pro 1565 1570 1575 Ile His Val Ser Pro Thr Phe Ala Ala Phe Val Glu Leu Pro Gly 1580 1585 1590 Thr His Gly Ile Thr His Gly Met Tyr Thr Ser Ala Ala Ala Arg 1595 1600 1605 Arg Phe Val Glu Thr Tyr Ala Ala Gln Asn Val Pro Glu Arg Val 1610 1615 1620 Lys His Tyr Glu Val Thr Phe Val Asn Met Val Leu Pro Asn Thr 1625 1630 1635 Glu Leu Ile Thr Lys Leu Ser His Thr Gly Met Ile Asn Gly Arg 1640 1645 1650 Lys Ile Ile Lys Val Glu Val Leu Asn Gln Glu Thr Ser Glu Pro 1655 1660 1665 Val Leu Val Gly Thr Ala Glu Val Glu Gln Pro Val Ser Ala Tyr 1670 1675 1680 Val Phe Thr Gly Gln Gly Ser Gln Glu Gln Gly Met Gly Met Asp 1685 1690 1695 Leu Tyr Ala Ser Ser Pro Val Ala Arg Lys Ile Trp Asp Ser Ala 1700 1705 1710 Asp Lys His Phe Leu Thr Asn Tyr Gly Phe Ser Ile Ile Asp Ile 1715 1720 1725 Val Lys His Asn Pro His Ser Ile Thr Ile His Phe Gly Gly Ser 1730 1735 1740 Lys Gly Lys Lys Ile Arg Asp Asn Tyr Met Ala Met Ala Tyr Glu 1745 1750 1755 Lys Leu Met Glu Asp Gly Thr Ser Lys Val Val Pro Val Phe Glu 1760 1765 1770 Thr Ile Thr Lys Asp Ser Thr Ser Phe Ser Phe Thr His Pro Ser 1775 1780 1785 Gly Leu Leu Ser Ala Thr Gln Phe Thr Gln Pro Ala Leu Thr Leu 1790 1795 1800 Met Glu Lys Ser Ala Phe Glu Asp Met Arg Ser Lys Gly Leu Val 1805 1810 1815 Gln Asn Asp Cys Ala Phe Ala Gly His Ser Leu Gly Glu Tyr Ser 1820 1825 1830 Ala Leu Ser Ala Met Gly Asp Val Leu Ser Ile Glu Ala Leu Val 1835 1840 1845 Asp Leu Val Phe Leu Arg Gly Leu Thr Met Gln Asn Ala Val His 1850 1855 1860 Arg Asp Glu Leu Gly Arg Ser Asp Tyr Gly Met Val Ala Ala Asn 1865 1870 1875 Pro Ser Arg Val Ser Ala Ser Phe Thr Asp Ala Ala Leu Arg Phe 1880 1885 1890 Ile Val Asp His Ile Gly Gln Gln Thr Asn Leu Leu Leu Glu Ile 1895 1900 1905 Val Asn Tyr Asn Val Glu Asn Gln Gln Tyr Val Val Ser Gly Asn 1910 1915 1920 Leu Leu Ser Leu Ser Thr Leu Gly His Val Leu Asn Phe Leu Lys 1925 1930 1935 Val Gln Lys Ile Asp Phe Glu Lys Leu Lys Glu Thr Leu Thr Ile 1940 1945 1950 Glu Gln Leu Lys Glu Gln Leu Thr Asp Ile Val Glu Ala Cys His 1955 1960 1965 Ala Lys Thr Leu Glu Gln Gln Lys Lys Thr Gly Arg Ile Glu Leu 1970 1975 1980 Glu Arg Gly Tyr Ala Thr Ile Pro Leu Lys Ile Asp Val Pro Phe 1985 1990 1995 His Ser Ser Phe Leu Arg Gly Gly Val Arg Met Phe Arg Glu Tyr 2000 2005 2010 Leu Val Lys Lys Ile Phe Pro His Gln Ile Asn Val Ala Lys Leu 2015 2020 2025 Arg Gly Lys Tyr Ile Pro Asn Leu Thr Ala Lys Pro Phe Glu Ile 2030 2035 2040 Ser Lys Glu Tyr Phe Gln Asn Val Tyr Asp Leu Thr Gly Ser Gln 2045 2050 2055 Arg Ile Lys Lys Ile Leu Gln Asn Trp Asp Glu Tyr Glu Ser Ser 2060 2065 2070 16 6232 DNA Schizosaccharomyces pombe 16 aagcttacta tactgtgtag tagagagtga taaaatgtta attatgccac aagcagttgc 60 taattcgcta tatttgataa cgatgcgtta aatattcgtt acatgcttca aagctgatag 120 gtttaacctg agtgttccca cgcgatagta aaggatcaag ttaactagaa ccaacaacta 180 aagcaggtgt tggagttttg ttaaaccatt tgaataatga gaccagaagt tgagcaggag 240 cttgctcata ctttattatt ggagttgctt gcataccagt ttgcatctcc tgtccgttgg 300 attgagacgc aagatgtaat tctttctcct ccagtatcgg ctgaacgtat cgtcgaaatt 360 ggacctagtc ctaccttagc tggtatggct aagcgtacct tgaaattgaa atatgagaac 420 atggatgccg ctttaagtat taatcgtgaa gttctttgct actctaaaga tgctcgtgaa 480 atctattaca actttgagga cgaggttgct gatgaacctg ccgaagcccc agcttcaacc 540 agctccactc caaaggttga aactgctgct gctgccgctc ccgctgccac gccagcccct 600 gccccagcac aaacatcagc cccagctgct gctttacctg acgagcctcc caaagctctt 660 gaggtacttc atactcttgt tgcccaaaag ttgaagaaaa gcatcgagga agtctcccct 720 caaaaatcta tcaaagattt ggttggcggt aagtccactt tgcaaaacga aattcttggt 780 gatttacaga aggagttcgg tgccactccc gagaagccag aggaggttcc attggatgag 840 cttggagcta tcatgcagtc aagctttaac ggatctcttg gtaaacaatc gtcttctctt 900 atctcacgaa tgatttcctc aaaaatgcct ggtggtttca ataattctgc tgttcgtggt 960 tatttaggaa accgttatgg tttgggtcct ggtcgtttgg agtctgtgct tttgttagcg 1020 cttaccatgg aacctgcatc acgtttgggc tcggaagctg atgctaaagc ttggcttgat 1080 agtgtagctc aaaaatatgc tgctcgtaat ggtgttacat tatcttctcc tactgctgaa 1140 ggcggttctt cgtccggttc tgcagctgtt atcgatgaag aaacctttaa gaaactcacc 1200 aagaataata ccatgcttgt tactcagcaa ttagaactat ttgctcgata cctcaataaa 1260 gaccttcgtg ctggccaaaa ggctcaagtt gctgaaaagg ttatttccga taccttacgc 1320 gctcaattag atttatggaa cgaagaacat ggtgaatttt atgcatcagg aattgctcct 1380 attttttcgc ctttaaaagc tcgcgtttac gactccgact ggaattgggc tcgtcaagat 1440 gctcttaaga tgttttttga cattatcttt ggtcgtctta ggcatgttga tactgaaata 1500 gtcgctcgtt gtatttctgt tatgaataga tccaacccta ctttacttga atttatgcaa 1560 tatcatattg atcattgtcc cgccgaaaag ggtgaaacat atcaacttgc taaaaccttg 1620 ggccaacagc taattgataa ttgcaaatcc gtgatagatg ctcctccagt tttcaaaaat 1680 gtgaatcatc caactgctcc ttctacgacg attgacgaac gtggtaattt gaattatgaa 1740 gaaatcccta gaccaggtgt tcgcaaatta actcattacg ttactgagat ggccaaaggt 1800 ggtaaattac caacggagtc caaaaacaaa gctaaggtac aaaacgattt ggctcgaatt 1860 tatcgcatta ttaagtctca aaacaaaatg tctcgttcgt ctaagttgca gattaaacag 1920 ttgtacggtc aggttttaca tgccctttcc cttccattgc cttcttccaa cgatgaacaa 1980 acgcctgtta aagaaaccat tcctttcctt catattagga agaagtccgt tgatggtaat 2040 tgggaattca acaagtcatt gactggcact tatttagatg ttttagaatc gggtgctaag 2100 aatggtataa cataccaaga caaatatgct ctagtgactg gtgcaggtgc aggctccatt 2160 ggtgctcaga ttgttgaagg tctccttgct ggtggtgcta aagttgtagt tactacatcc 2220 cggttttcgc gcaaggttac tgaattttat caatcccttt acacccgcca tggaagccgt 2280 ggttcatgtc tgatcgtggt tccatttaac caaggatcta agacagacgt agaagctctt 2340 attgattata tttatgacga aaagaagggt cttggatgga acttggacta cattgttcct 2400 ttcgctgcca ttccagaaaa tggtcgtgaa attgatggca ttgattctcg ttccgagttt 2460 gctcaccgta ttatgttgac aaacattttg agactgcttg gcgccgtcaa aagtcaaaag 2520 gcctctcgtg gtatggatac ccgacccgct caagttattt tgcctctttc tcccaatcac 2580 ggtacctttg gaaacgatgg tttatactcg gaatctaagt taggtttaga aactttgttt 2640 aaccgttggt actccgagtc atgggctaat tacctaacca tttgtggggc tgtcattggt 2700 tggactcgtg gtacaggctt aatggcacct aataatattg tttctcaggg aatcgaaaaa 2760 tatggtgttc gtactttttc gcagagtgag atggctttta acattttggg tttgatgtcc 2820 cagaaagtcg tcgacttgtg tcaatctgaa ccaatttatg ccaaccttaa cggtggtctt 2880 gagcttttac ctgatctcaa ggacctttcc actcgtttgc gtaccgaatt gttagaaact 2940 gccgaaatcc gccgcgctgt tgccgcagag actgcctttg atcatagcat taccaacgga 3000 cctgactctg aagcagtttt ccagaaaact gccattcagc ctagggccaa tcttaaattt 3060 aatttcccca aattgaaacc ttatgaagcc ctttctcatt tatctgatct tcgtggaatg 3120 gttgatttag aaaaagttcc tgttgttact ggtttttccg aagtaggtcc atggggtaac 3180 tctcgtacta gatgggatat ggagtgttat ggtgagtttt cactagaagg atgtgtcgaa 3240 attgcttgga ttatgggatt aattaaaaac ttcaatggca agggcaaaga cggcaagccc 3300 tattcaggtt gggttgatac aaagaccggt gaacctgtgg acgacaaaga cgttaaagct 3360 aagtatgaga agtatatact ggagcattgc ggtatccgta ttattgaagc tgaactcttc 3420 catggatata atcctgaaaa gaaagagctt ttgcaagaag ttgttattga tcatgactta 3480 gagccttttg aagcatccaa agaggctgct catgagttca agcttcgtca tggtgatcaa 3540 gttgaaattt ttgaaattcc tgattctacc gaatggtccg tacgcttcaa gcgcggtaca 3600 agtatgctaa ttcctaaggc tttgcgcttt gatcgatttg ttgctggcca gattccactt 3660 ggttgggatc ccaaacgtta tggcattcct gacgatatta tttctcaagt tgaccctaca 3720 actttgtacg ttttagtgtc tactgtagaa gctctggttg catcaggtat tacagatcct 3780 tatgaatgct ataagtatat tcacgtatct gaacttggta atacagttgg ttctggtatt 3840 ggtggtatgt ctgctcttcg tggaatgtac aaggaccgct ggactgataa acctgttcaa 3900 aaagatattt tacaagaatc attcattaac actgccaatg cttggattaa catgcttttg 3960 ctctctgcct ctggtcctat taagactcct gttggtgctt gcgctaccgc tgtcgaatct 4020 gttgatgcag ctgtcgactt gatcacttct ggtaaggcca ggatatgtat tagcggtggt 4080 tatgacgact tttcagaaga aggttcatac gagtttgcga acatgggtgc tacatcaaat 4140 gctgctaagg aaacagaaag gggacgtact cctcaagaaa tgtctcgtcc tgctacttct 4200 actcgtgatg gatttatgga gtctcaaggt gctggtgtac agattatcat gcaagcaaag 4260 cttgctattg agatgggtgt ccctatacat ggtattgttg gttatgtttc cacagctatg 4320 gataaacaag gtcgttcggt tcctgcccct gggcaaggta ttttgactgg tgctcgtgaa 4380 atcgcgacta agacacccct tcccatagtt gaccttaaat tccgttctcg tcaactccaa 4440 cgccgccgtt ctcaaattgg tgaatgggcc gaacgcgagt atctttattt agaagaagaa 4500 cttgatgcga tgaaggttca aaatcctgac ttggatttag aggcttaccg tatagagcgt 4560 atcaacgtta ttaaggagga ggttgttcga caagaaaagg aggcgctcaa tacttttgga 4620 aatgaatttt ggaaacgtga tcctactatt gctcctatcc gtggtgcatt agctgtttgg 4680 ggtcttacta ttgacgattt gggcgttgca tcattccatg gtacctctac caaagccaat 4740 gagaagaatg aatgcgatgt cattgacagt cagttaacac atctcggacg ctctaagggt 4800 aacgctgtgt acggtgtttt ccagaaatat ctcactggac atagcaaggg tggtgctgga 4860 gcttggatgc tcaacggagc tctccaaatt cttcgctctg ggtttgttcc gggtaatcgt 4920 aacgccgata acattgatga gtatctagca cgattcgacc gggttatgtt ccctagtgaa 4980 ggtatacaaa ctgatggcat aaaggcagca tctgttactg catttggttt tggacaagtt 5040 ggtggacaag ttatagttat ccatcctgat tacatttacg gtgtgattga tgaggctact 5100 tataatgctt acaaagctaa aactgctgct cgttataagg catcttatcg ttacacccac 5160 gatgcgctgg tttacaacaa tttggtccgc gccaaggatt ctcctcctta caccaaagaa 5220 caagagaaag ccgtttatct caatcctttg gcacgcgctt cgaagagcaa agctggcact 5280 tggactttcc ctgccacact gcctgctgaa tccgacattt ctaaaaccaa cgaaactaca 5340 cgtactctac aaagcctaac aacctcattg accaactcca atgaaaatgt tggcgtggat 5400 gttgaacttg tatcagcgat tagcattgat aatgagacct ttatagaaag gaattttact 5460 gataccgagc gaaagtactg ttttgcagct cctaatcccc aagctagctt tgccggacgt 5520 tggtcagcca aagaggctgt ctttaagtct ttgggtattt ccggtaaagg cgctgcagct 5580 ccattgaagg atatcgaaat tatttcttca gagtctggtg ctcctgaagt agttttgcac 5640 ggagaggctg cgaaggctgc aacgaccgcc ggtgtgaaga gtgtttccgt cagtatttcc 5700 cacgatgata atcaaagtgt cagtgttgct ttggctcaca agtaatttac gttatattgt 5760 ctttcaacat tggtatgcgg attttcgcat tcccttcaat cgtttgattt aatacactat 5820 ctttaatctt tttgtttacc tcaaatgctt tgaaatggtt atcgattttt gtagtcgtta 5880 tatacgcagt tagaaataaa ttacttttaa ccttataaat tattatgctc taaaaaaatg 5940 cagtatcatt aaatttaaac gaatgtcctt acacgtatga gtatttaaac tgatattgag 6000 ttatcttcat aaatttctga agccaggcag cggttgttgt ttcatcgaaa gaaatggggt 6060 tcatatatgc tgttgaagtg ttttgctcga acaaaatttc agttagatgc ttaatcactg 6120 tccaaccgta agcattcgga aaccgcctac gacaatcatg gtattgtgtg catcaccatc 6180 gagtctaaca gcaacctttc tccacgcaag cctatgccag tttttggcaa tg 6232 17 1842 PRT Schizosaccharomyces pombe 17 Met Arg Pro Glu Val Glu Gln Glu Leu Ala His Thr Leu Leu Leu Glu 1 5 10 15 Leu Leu Ala Tyr Gln Phe Ala Ser Pro Val Arg Trp Ile Glu Thr Gln 20 25 30 Asp Val Ile Leu Ser Pro Pro Val Ser Ala Glu Arg Ile Val Glu Ile 35 40 45 Gly Pro Ser Pro Thr Leu Ala Gly Met Ala Lys Arg Thr Leu Lys Leu 50 55 60 Lys Tyr Glu Asn Met Asp Ala Ala Leu Ser Ile Asn Arg Glu Val Leu 65 70 75 80 Cys Tyr Ser Lys Asp Ala Arg Glu Ile Tyr Tyr Asn Phe Glu Asp Glu 85 90 95 Val Ala Asp Glu Pro Ala Glu Ala Pro Ala Ser Thr Ser Ser Thr Pro 100 105 110 Lys Val Glu Thr Ala Ala Ala Ala Ala Pro Ala Ala Thr Pro Ala Pro 115 120 125 Ala Pro Ala Gln Thr Ser Ala Pro Ala Ala Ala Leu Pro Asp Glu Pro 130 135 140 Pro Lys Ala Leu Glu Val Leu His Thr Leu Val Ala Gln Lys Leu Lys 145 150 155 160 Lys Ser Ile Glu Glu Val Ser Pro Gln Lys Ser Ile Lys Asp Leu Val 165 170 175 Gly Gly Lys Ser Thr Leu Gln Asn Glu Ile Leu Gly Asp Leu Gln Lys 180 185 190 Glu Phe Gly Ala Thr Pro Glu Lys Pro Glu Glu Val Pro Leu Asp Glu 195 200 205 Leu Gly Ala Ile Met Gln Ser Ser Phe Asn Gly Ser Leu Gly Lys Gln 210 215 220 Ser Ser Ser Leu Ile Ser Arg Met Ile Ser Ser Lys Met Pro Gly Gly 225 230 235 240 Phe Asn Asn Ser Ala Val Arg Gly Tyr Leu Gly Asn Arg Tyr Gly Leu 245 250 255 Gly Pro Gly Arg Leu Glu Ser Val Leu Leu Leu Ala Leu Thr Met Glu 260 265 270 Pro Ala Ser Arg Leu Gly Ser Glu Ala Asp Ala Lys Ala Trp Leu Asp 275 280 285 Ser Val Ala Gln Lys Tyr Ala Ala Arg Asn Gly Val Thr Leu Ser Ser 290 295 300 Pro Thr Ala Glu Gly Gly Ser Ser Ser Gly Ser Ala Ala Val Ile Asp 305 310 315 320 Glu Glu Thr Phe Lys Lys Leu Thr Lys Asn Asn Thr Met Leu Val Thr 325 330 335 Gln Gln Leu Glu Leu Phe Ala Arg Tyr Leu Asn Lys Asp Leu Arg Ala 340 345 350 Gly Gln Lys Ala Gln Val Ala Glu Lys Val Ile Ser Asp Thr Leu Arg 355 360 365 Ala Gln Leu Asp Leu Trp Asn Glu Glu His Gly Glu Phe Tyr Ala Ser 370 375 380 Gly Ile Ala Pro Ile Phe Ser Pro Leu Lys Ala Arg Val Tyr Asp Ser 385 390 395 400 Asp Trp Asn Trp Ala Arg Gln Asp Ala Leu Lys Met Phe Phe Asp Ile 405 410 415 Ile Phe Gly Arg Leu Arg His Val Asp Thr Glu Ile Val Ala Arg Cys 420 425 430 Ile Ser Val Met Asn Arg Ser Asn Pro Thr Leu Leu Glu Phe Met Gln 435 440 445 Tyr His Ile Asp His Cys Pro Ala Glu Lys Gly Glu Thr Tyr Gln Leu 450 455 460 Ala Lys Thr Leu Gly Gln Gln Leu Ile Asp Asn Cys Lys Ser Val Ile 465 470 475 480 Asp Ala Pro Pro Val Phe Lys Asn Val Asn His Pro Thr Ala Pro Ser 485 490 495 Thr Thr Ile Asp Glu Arg Gly Asn Leu Asn Tyr Glu Glu Ile Pro Arg 500 505 510 Pro Gly Val Arg Lys Leu Thr His Tyr Val Thr Glu Met Ala Lys Gly 515 520 525 Gly Lys Leu Pro Thr Glu Ser Lys Asn Lys Ala Lys Val Gln Asn Asp 530 535 540 Leu Ala Arg Ile Tyr Arg Ile Ile Lys Ser Gln Asn Lys Met Ser Arg 545 550 555 560 Ser Ser Lys Leu Gln Ile Lys Gln Leu Tyr Gly Gln Val Leu His Ala 565 570 575 Leu Ser Leu Pro Leu Pro Ser Ser Asn Asp Glu Gln Thr Pro Val Lys 580 585 590 Glu Thr Ile Pro Phe Leu His Ile Arg Lys Lys Ser Val Asp Gly Asn 595 600 605 Trp Glu Phe Asn Lys Ser Leu Thr Gly Thr Tyr Leu Asp Val Leu Glu 610 615 620 Ser Gly Ala Lys Asn Gly Ile Thr Tyr Gln Asp Lys Tyr Ala Leu Val 625 630 635 640 Thr Gly Ala Gly Ala Gly Ser Ile Gly Ala Gln Ile Val Glu Gly Leu 645 650 655 Leu Ala Gly Gly Ala Lys Val Val Val Thr Thr Ser Arg Phe Ser Arg 660 665 670 Lys Val Thr Glu Phe Tyr Gln Ser Leu Tyr Thr Arg His Gly Ser Arg 675 680 685 Gly Ser Cys Leu Ile Val Val Pro Phe Asn Gln Gly Ser Lys Thr Asp 690 695 700 Val Glu Ala Leu Ile Asp Tyr Ile Tyr Asp Glu Lys Lys Gly Leu Gly 705 710 715 720 Trp Asn Leu Asp Tyr Ile Val Pro Phe Ala Ala Ile Pro Glu Asn Gly 725 730 735 Arg Glu Ile Asp Gly Ile Asp Ser Arg Ser Glu Phe Ala His Arg Ile 740 745 750 Met Leu Thr Asn Ile Leu Arg Leu Leu Gly Ala Val Lys Ser Gln Lys 755 760 765 Ala Ser Arg Gly Met Asp Thr Arg Pro Ala Gln Val Ile Leu Pro Leu 770 775 780 Ser Pro Asn His Gly Thr Phe Gly Asn Asp Gly Leu Tyr Ser Glu Ser 785 790 795 800 Lys Leu Gly Leu Glu Thr Leu Phe Asn Arg Trp Tyr Ser Glu Ser Trp 805 810 815 Ala Asn Tyr Leu Thr Ile Cys Gly Ala Val Ile Gly Trp Thr Arg Gly 820 825 830 Thr Gly Leu Met Ala Pro Asn Asn Ile Val Ser Gln Gly Ile Glu Lys 835 840 845 Tyr Gly Val Arg Thr Phe Ser Gln Ser Glu Met Ala Phe Asn Ile Leu 850 855 860 Gly Leu Met Ser Gln Lys Val Val Asp Leu Cys Gln Ser Glu Pro Ile 865 870 875 880 Tyr Ala Asn Leu Asn Gly Gly Leu Glu Leu Leu Pro Asp Leu Lys Asp 885 890 895 Leu Ser Thr Arg Leu Arg Thr Glu Leu Leu Glu Thr Ala Glu Ile Arg 900 905 910 Arg Ala Val Ala Ala Glu Thr Ala Phe Asp His Ser Ile Thr Asn Gly 915 920 925 Pro Asp Ser Glu Ala Val Phe Gln Lys Thr Ala Ile Gln Pro Arg Ala 930 935 940 Asn Leu Lys Phe Asn Phe Pro Lys Leu Lys Pro Tyr Glu Ala Leu Ser 945 950 955 960 His Leu Ser Asp Leu Arg Gly Met Val Asp Leu Glu Lys Val Pro Val 965 970 975 Val Thr Gly Phe Ser Glu Val Gly Pro Trp Gly Asn Ser Arg Thr Arg 980 985 990 Trp Asp Met Glu Cys Tyr Gly Glu Phe Ser Leu Glu Gly Cys Val Glu 995 1000 1005 Ile Ala Trp Ile Met Gly Leu Ile Lys Asn Phe Asn Gly Lys Gly 1010 1015 1020 Lys Asp Gly Lys Pro Tyr Ser Gly Trp Val Asp Thr Lys Thr Gly 1025 1030 1035 Glu Pro Val Asp Asp Lys Asp Val Lys Ala Lys Tyr Glu Lys Tyr 1040 1045 1050 Ile Leu Glu His Cys Gly Ile Arg Ile Ile Glu Ala Glu Leu Phe 1055 1060 1065 His Gly Tyr Asn Pro Glu Lys Lys Glu Leu Leu Gln Glu Val Val 1070 1075 1080 Ile Asp His Asp Leu Glu Pro Phe Glu Ala Ser Lys Glu Ala Ala 1085 1090 1095 His Glu Phe Lys Leu Arg His Gly Asp Gln Val Glu Ile Phe Glu 1100 1105 1110 Ile Pro Asp Ser Thr Glu Trp Ser Val Arg Phe Lys Arg Gly Thr 1115 1120 1125 Ser Met Leu Ile Pro Lys Ala Leu Arg Phe Asp Arg Phe Val Ala 1130 1135 1140 Gly Gln Ile Pro Leu Gly Trp Asp Pro Lys Arg Tyr Gly Ile Pro 1145 1150 1155 Asp Asp Ile Ile Ser Gln Val Asp Pro Thr Thr Leu Tyr Val Leu 1160 1165 1170 Val Ser Thr Val Glu Ala Leu Val Ala Ser Gly Ile Thr Asp Pro 1175 1180 1185 Tyr Glu Cys Tyr Lys Tyr Ile His Val Ser Glu Leu Gly Asn Thr 1190 1195 1200 Val Gly Ser Gly Ile Gly Gly Met Ser Ala Leu Arg Gly Met Tyr 1205 1210 1215 Lys Asp Arg Trp Thr Asp Lys Pro Val Gln Lys Asp Ile Leu Gln 1220 1225 1230 Glu Ser Phe Ile Asn Thr Ala Asn Ala Trp Ile Asn Met Leu Leu 1235 1240 1245 Leu Ser Ala Ser Gly Pro Ile Lys Thr Pro Val Gly Ala Cys Ala 1250 1255 1260 Thr Ala Val Glu Ser Val Asp Ala Ala Val Asp Leu Ile Thr Ser 1265 1270 1275 Gly Lys Ala Arg Ile Cys Ile Ser Gly Gly Tyr Asp Asp Phe Ser 1280 1285 1290 Glu Glu Gly Ser Tyr Glu Phe Ala Asn Met Gly Ala Thr Ser Asn 1295 1300 1305 Ala Ala Lys Glu Thr Glu Arg Gly Arg Thr Pro Gln Glu Met Ser 1310 1315 1320 Arg Pro Ala Thr Ser Thr Arg Asp Gly Phe Met Glu Ser Gln Gly 1325 1330 1335 Ala Gly Val Gln Ile Ile Met Gln Ala Lys Leu Ala Ile Glu Met 1340 1345 1350 Gly Val Pro Ile His Gly Ile Val Gly Tyr Val Ser Thr Ala Met 1355 1360 1365 Asp Lys Gln Gly Arg Ser Val Pro Ala Pro Gly Gln Gly Ile Leu 1370 1375 1380 Thr Gly Ala Arg Glu Ile Ala Thr Lys Thr Pro Leu Pro Ile Val 1385 1390 1395 Asp Leu Lys Phe Arg Ser Arg Gln Leu Gln Arg Arg Arg Ser Gln 1400 1405 1410 Ile Gly Glu Trp Ala Glu Arg Glu Tyr Leu Tyr Leu Glu Glu Glu 1415 1420 1425 Leu Asp Ala Met Lys Val Gln Asn Pro Asp Leu Asp Leu Glu Ala 1430 1435 1440 Tyr Arg Ile Glu Arg Ile Asn Val Ile Lys Glu Glu Val Val Arg 1445 1450 1455 Gln Glu Lys Glu Ala Leu Asn Thr Phe Gly Asn Glu Phe Trp Lys 1460 1465 1470 Arg Asp Pro Thr Ile Ala Pro Ile Arg Gly Ala Leu Ala Val Trp 1475 1480 1485 Gly Leu Thr Ile Asp Asp Leu Gly Val Ala Ser Phe His Gly Thr 1490 1495 1500 Ser Thr Lys Ala Asn Glu Lys Asn Glu Cys Asp Val Ile Asp Ser 1505 1510 1515 Gln Leu Thr His Leu Gly Arg Ser Lys Gly Asn Ala Val Tyr Gly 1520 1525 1530 Val Phe Gln Lys Tyr Leu Thr Gly His Ser Lys Gly Gly Ala Gly 1535 1540 1545 Ala Trp Met Leu Asn Gly Ala Leu Gln Ile Leu Arg Ser Gly Phe 1550 1555 1560 Val Pro Gly Asn Arg Asn Ala Asp Asn Ile Asp Glu Tyr Leu Ala 1565 1570 1575 Arg Phe Asp Arg Val Met Phe Pro Ser Glu Gly Ile Gln Thr Asp 1580 1585 1590 Gly Ile Lys Ala Ala Ser Val Thr Ala Phe Gly Phe Gly Gln Val 1595 1600 1605 Gly Gly Gln Val Ile Val Ile His Pro Asp Tyr Ile Tyr Gly Val 1610 1615 1620 Ile Asp Glu Ala Thr Tyr Asn Ala Tyr Lys Ala Lys Thr Ala Ala 1625 1630 1635 Arg Tyr Lys Ala Ser Tyr Arg Tyr Thr His Asp Ala Leu Val Tyr 1640 1645 1650 Asn Asn Leu Val Arg Ala Lys Asp Ser Pro Pro Tyr Thr Lys Glu 1655 1660 1665 Gln Glu Lys Ala Val Tyr Leu Asn Pro Leu Ala Arg Ala Ser Lys 1670 1675 1680 Ser Lys Ala Gly Thr Trp Thr Phe Pro Ala Thr Leu Pro Ala Glu 1685 1690 1695 Ser Asp Ile Ser Lys Thr Asn Glu Thr Thr Arg Thr Leu Gln Ser 1700 1705 1710 Leu Thr Thr Ser Leu Thr Asn Ser Asn Glu Asn Val Gly Val Asp 1715 1720 1725 Val Glu Leu Val Ser Ala Ile Ser Ile Asp Asn Glu Thr Phe Ile 1730 1735 1740 Glu Arg Asn Phe Thr Asp Thr Glu Arg Lys Tyr Cys Phe Ala Ala 1745 1750 1755 Pro Asn Pro Gln Ala Ser Phe Ala Gly Arg Trp Ser Ala Lys Glu 1760 1765 1770 Ala Val Phe Lys Ser Leu Gly Ile Ser Gly Lys Gly Ala Ala Ala 1775 1780 1785 Pro Leu Lys Asp Ile Glu Ile Ile Ser Ser Glu Ser Gly Ala Pro 1790 1795 1800 Glu Val Val Leu His Gly Glu Ala Ala Lys Ala Ala Thr Thr Ala 1805 1810 1815 Gly Val Lys Ser Val Ser Val Ser Ile Ser His Asp Asp Asn Gln 1820 1825 1830 Ser Val Ser Val Ala Leu Ala His Lys 1835 1840 18 2051 PRT Saccharomyces cerevisiae 18 Met Asp Ala Tyr Ser Thr Arg Pro Leu Thr Leu Ser His Gly Ser Leu 1 5 10 15 Glu His Val Leu Leu Val Pro Thr Ala Ser Phe Phe Ile Ala Ser Gln 20 25 30 Leu Gln Glu Gln Phe Asn Lys Ile Leu Pro Glu Pro Thr Glu Gly Phe 35 40 45 Ala Ala Asp Asp Glu Pro Thr Thr Pro Ala Glu Leu Val Gly Lys Phe 50 55 60 Leu Gly Tyr Val Ser Ser Leu Val Glu Pro Ser Lys Val Gly Gln Phe 65 70 75 80 Asp Gln Val Leu Asn Leu Cys Leu Thr Glu Phe Glu Asn Cys Tyr Leu 85 90 95 Glu Gly Asn Asp Ile His Ala Leu Ala Ala Lys Leu Leu Gln Glu Asn 100 105 110 Asp Thr Thr Leu Val Lys Thr Lys Glu Leu Ile Lys Asn Tyr Ile Thr 115 120 125 Ala Arg Ile Met Ala Lys Arg Pro Phe Asp Lys Lys Ser Asn Ser Ala 130 135 140 Leu Phe Arg Ala Val Gly Glu Gly Asn Ala Gln Leu Val Ala Ile Phe 145 150 155 160 Gly Gly Gln Gly Asn Thr Asp Asp Tyr Phe Glu Glu Leu Arg Asp Leu 165 170 175 Tyr Gln Thr Tyr His Val Leu Val Gly Asp Leu Ile Lys Phe Ser Ala 180 185 190 Glu Thr Leu Ser Glu Leu Ile Arg Thr Thr Leu Asp Ala Glu Lys Val 195 200 205 Phe Thr Gln Gly Leu Asn Ile Leu Glu Trp Leu Glu Asn Pro Ser Asn 210 215 220 Thr Pro Asp Lys Asp Tyr Leu Leu Ser Ile Pro Ile Ser Cys Pro Leu 225 230 235 240 Ile Gly Val Ile Gln Leu Ala His Tyr Val Val Thr Ala Lys Leu Leu 245 250 255 Gly Phe Thr Pro Gly Glu Leu Arg Ser Tyr Leu Lys Gly Ala Thr Gly 260 265 270 His Ser Gln Gly Leu Val Thr Ala Val Ala Ile Ala Glu Thr Asp Ser 275 280 285 Trp Glu Ser Phe Phe Val Ser Val Arg Lys Ala Ile Thr Val Leu Phe 290 295 300 Phe Ile Gly Val Arg Cys Tyr Glu Ala Tyr Pro Asn Thr Ser Leu Pro 305 310 315 320 Pro Ser Ile Leu Glu Asp Ser Leu Glu Asn Asn Glu Gly Val Pro Ser 325 330 335 Pro Met Leu Ser Ile Ser Asn Leu Thr Gln Glu Gln Val Gln Asp Tyr 340 345 350 Val Asn Lys Thr Asn Ser His Leu Pro Ala Gly Lys Gln Val Glu Ile 355 360 365 Ser Leu Val Asn Gly Ala Lys Asn Leu Val Val Ser Gly Pro Pro Gln 370 375 380 Ser Leu Tyr Gly Leu Asn Leu Thr Leu Arg Lys Ala Lys Ala Pro Ser 385 390 395 400 Gly Leu Asp Gln Ser Arg Ile Pro Phe Ser Glu Arg Lys Leu Lys Phe 405 410 415 Ser Asn Arg Phe Leu Pro Val Ala Ser Pro Phe His Ser His Leu Leu 420 425 430 Val Pro Ala Ser Asp Leu Ile Asn Lys Asp Leu Val Lys Asn Asn Val 435 440 445 Ser Phe Asn Ala Lys Asp Ile Gln Ile Pro Val Tyr Asp Thr Phe Asp 450 455 460 Gly Ser Asp Leu Arg Val Leu Ser Gly Ser Ile Ser Glu Arg Ile Val 465 470 475 480 Asp Cys Ile Ile Arg Leu Pro Val Lys Trp Glu Thr Thr Thr Gln Phe 485 490 495 Lys Ala Thr His Ile Leu Asp Phe Gly Pro Gly Gly Ala Ser Gly Leu 500 505 510 Gly Val Leu Thr His Arg Asn Lys Asp Gly Thr Gly Val Arg Val Ile 515 520 525 Val Ala Gly Thr Leu Asp Ile Asn Pro Asp Asp Asp Tyr Gly Phe Lys 530 535 540 Gln Glu Ile Phe Asp Val Thr Ser Asn Gly Leu Lys Lys Asn Pro Asn 545 550 555 560 Trp Leu Glu Glu Tyr His Pro Lys Leu Ile Lys Asn Lys Ser Gly Lys 565 570 575 Ile Phe Val Glu Thr Lys Phe Ser Lys Leu Ile Gly Arg Pro Pro Leu 580 585 590 Leu Val Pro Gly Met Thr Pro Cys Thr Val Ser Pro Asp Phe Val Ala 595 600 605 Ala Thr Thr Asn Ala Gly Tyr Thr Ile Glu Leu Ala Gly Gly Gly Tyr 610 615 620 Phe Ser Ala Ala Gly Met Thr Ala Ala Ile Asp Ser Val Val Ser Gln 625 630 635 640 Ile Glu Lys Gly Ser Thr Phe Gly Ile Asn Leu Ile Tyr Val Asn Pro 645 650 655 Phe Met Leu Gln Trp Gly Ile Pro Leu Ile Lys Glu Leu Arg Ser Lys 660 665 670 Gly Tyr Pro Ile Gln Phe Leu Thr Ile Gly Ala Gly Val Pro Ser Leu 675 680 685 Glu Val Ala Ser Glu Tyr Ile Glu Thr Leu Gly Leu Lys Tyr Leu Gly 690 695 700 Leu Lys Pro Gly Ser Ile Asp Ala Ile Ser Gln Val Ile Asn Ile Ala 705 710 715 720 Lys Ala His Pro Asn Phe Pro Ile Ala Leu Gln Trp Thr Gly Gly Arg 725 730 735 Gly Gly Gly His His Ser Phe Glu Asp Ala His Thr Pro Met Leu Gln 740 745 750 Met Tyr Ser Lys Ile Arg Arg His Pro Asn Ile Met Leu Ile Phe Gly 755 760 765 Ser Gly Phe Gly Ser Ala Asp Asp Thr Tyr Pro Tyr Leu Thr Gly Glu 770 775 780 Trp Ser Thr Lys Phe Asp Tyr Pro Pro Met Pro Phe Asp Gly Phe Leu 785 790 795 800 Phe Gly Ser Arg Val Met Ile Ala Lys Glu Val Lys Thr Ser Pro Asp 805 810 815 Ala Lys Lys Cys Ile Ala Ala Cys Thr Gly Val Pro Asp Asp Lys Trp 820 825 830 Glu Gln Thr Tyr Lys Lys Pro Thr Gly Gly Ile Val Thr Val Arg Ser 835 840 845 Glu Met Gly Glu Pro Ile His Lys Ile Ala Thr Arg Gly Val Met Leu 850 855 860 Trp Lys Glu Phe Asp Glu Thr Ile Phe Asn Leu Pro Lys Asn Lys Leu 865 870 875 880 Val Pro Thr Leu Glu Ala Lys Arg Asp Tyr Ile Ile Ser Arg Leu Asn 885 890 895 Ala Asp Phe Gln Lys Pro Trp Phe Ala Thr Val Asn Gly Gln Ala Arg 900 905 910 Asp Leu Ala Thr Met Thr Tyr Glu Glu Val Ala Lys Arg Leu Val Glu 915 920 925 Leu Met Phe Ile Arg Ser Thr Asn Ser Trp Phe Asp Val Thr Trp Arg 930 935 940 Thr Phe Thr Gly Asp Phe Leu Arg Arg Val Glu Glu Arg Phe Thr Lys 945 950 955 960 Ser Lys Thr Leu Ser Leu Ile Gln Ser Tyr Ser Leu Leu Asp Lys Pro 965 970 975 Asp Glu Ala Ile Glu Lys Val Phe Asn Ala Tyr Pro Ala Ala Arg Glu 980 985 990 Gln Phe Leu Asn Ala Gln Asp Ile Asp His Phe Leu Ser Met Cys Gln 995 1000 1005 Asn Pro Met Gln Lys Pro Val Pro Phe Val Pro Val Leu Asp Arg 1010 1015 1020 Arg Phe Glu Ile Phe Phe Lys Lys Asp Ser Leu Trp Gln Ser Glu 1025 1030 1035 His Leu Glu Ala Val Val Asp Gln Asp Val Gln Arg Thr Cys Ile 1040 1045 1050 Leu His Gly Pro Val Ala Ala Gln Phe Thr Lys Val Ile Asp Glu 1055 1060 1065 Pro Ile Lys Ser Ile Met Asp Gly Ile His Asp Gly His Ile Lys 1070 1075 1080 Lys Leu Leu His Gln Tyr Tyr Gly Asp Asp Glu Ser Lys Ile Pro 1085 1090 1095 Ala Val Glu Tyr Phe Gly Gly Glu Ser Pro Val Asp Val Gln Ser 1100 1105 1110 Gln Val Asp Ser Ser Ser Val Ser Glu Asp Ser Ala Val Phe Lys 1115 1120 1125 Ala Thr Ser Ser Thr Asp Glu Glu Ser Trp Phe Lys Ala Leu Ala 1130 1135 1140 Gly Ser Glu Ile Asn Trp Arg His Ala Ser Phe Leu Cys Ser Phe 1145 1150 1155 Ile Thr Gln Asp Lys Met Phe Val Ser Asn Pro Ile Arg Lys Val 1160 1165 1170 Phe Lys Pro Ser Gln Gly Met Val Val Glu Ile Ser Asn Gly Asn 1175 1180 1185 Thr Ser Ser Lys Thr Val Val Thr Leu Ser Glu Pro Val Gln Gly 1190 1195 1200 Glu Leu Lys Pro Thr Val Ile Leu Lys Leu Leu Lys Glu Asn Ile 1205 1210 1215 Ile Gln Met Glu Met Ile Glu Asn Arg Thr Met Asp Gly Lys Pro 1220 1225 1230 Val Ser Leu Pro Leu Leu Tyr Asn Phe Asn Pro Asp Asn Gly Phe 1235 1240 1245 Ala Pro Ile Ser Glu Val Met Glu Asp Arg Asn Gln Arg Ile Lys 1250 1255 1260 Glu Met Tyr Trp Lys Leu Trp Ile Asp Glu Pro Phe Asn Leu Asp 1265 1270 1275 Phe Asp Pro Arg Asp Val Ile Lys Gly Lys Asp Phe Glu Ile Thr 1280 1285 1290 Ala Lys Glu Val Tyr Asp Phe Thr His Ala Val Gly Asn Asn Cys 1295 1300 1305 Glu Asp Phe Val Ser Arg Pro Asp Arg Thr Met Leu Ala Pro Met 1310 1315 1320 Asp Phe Ala Ile Val Val Gly Trp Arg Ala Ile Ile Lys Ala Ile 1325 1330 1335 Phe Pro Asn Thr Val Asp Gly Asp Leu Leu Lys Leu Val His Leu 1340 1345 1350 Ser Asn Gly Tyr Lys Met Ile Pro Gly Ala Lys Pro Leu Gln Val 1355 1360 1365 Gly Asp Val Val Ser Thr Thr Ala Val Ile Glu Ser Val Val Asn 1370 1375 1380 Gln Pro Thr Gly Lys Ile Val Asp Val Val Gly Thr Leu Ser Arg 1385 1390 1395 Asn Gly Lys Pro Val Met Glu Val Thr Ser Ser Phe Phe Tyr Arg 1400 1405 1410 Gly Asn Tyr Thr Asp Phe Glu Asn Thr Phe Gln Lys Thr Val Glu 1415 1420 1425 Pro Val Tyr Gln Met His Ile Lys Thr Ser Lys Asp Ile Ala Val 1430 1435 1440 Leu Arg Ser Lys Glu Trp Phe Gln Leu Asp Asp Glu Asp Phe Asp 1445 1450 1455 Leu Leu Asn Lys Thr Leu Thr Phe Glu Thr Glu Thr Glu Val Thr 1460 1465 1470 Phe Lys Asn Ala Asn Ile Phe Ser Ser Val Lys Cys Phe Gly Pro 1475 1480 1485 Ile Lys Val Glu Leu Pro Thr Lys Glu Thr Val Glu Ile Gly Ile 1490 1495 1500 Val Asp Tyr Glu Ala Gly Ala Ser His Gly Asn Pro Val Val Asp 1505 1510 1515 Phe Leu Lys Arg Asn Gly Ser Thr Leu Glu Gln Lys Val Asn Leu 1520 1525 1530 Glu Asn Pro Ile Pro Ile Ala Val Leu Asp Ser Tyr Thr Pro Ser 1535 1540 1545 Thr Asn Glu Pro Tyr Ala Arg Val Ser Gly Asp Leu Asn Pro Ile 1550 1555 1560 His Val Ser Arg His Phe Ala Ser Tyr Ala Asn Leu Pro Gly Thr 1565 1570 1575 Ile Thr His Gly Met Phe Ser Ser Ala Ser Val Arg Ala Leu Ile 1580 1585 1590 Glu Asn Trp Ala Ala Asp Ser Val Ser Ser Arg Val Arg Gly Tyr 1595 1600 1605 Thr Cys Gln Phe Val Asp Met Val Leu Pro Asn Thr Ala Leu Lys 1610 1615 1620 Thr Ser Ile Gln His Val Gly Met Ile Asn Gly Arg Lys Leu Ile 1625 1630 1635 Lys Phe Glu Thr Arg Asn Glu Asp Asp Val Val Val Leu Thr Gly 1640 1645 1650 Glu Ala Glu Ile Glu Gln Pro Val Thr Thr Phe Val Phe Thr Gly 1655 1660 1665 Gln Gly Ser Gln Glu Gln Gly Met Gly Met Asp Leu Tyr Lys Thr 1670 1675 1680 Ser Lys Ala Ala Gln Asp Val Trp Asn Arg Ala Asp Asn His Phe 1685 1690 1695 Lys Asp Thr Tyr Gly Phe Ser Ile Leu Asp Ile Val Ile Asn Asn 1700 1705 1710 Pro Val Asn Leu Thr Ile His Phe Gly Gly Glu Lys Gly Lys Arg 1715 1720 1725 Ile Arg Glu Asn Tyr Ser Ala Met Ile Phe Glu Thr Ile Val Asp 1730 1735 1740 Gly Lys Leu Lys Thr Glu Lys Ile Phe Lys Glu Ile Asn Glu His 1745 1750 1755 Ser Thr Ser Tyr Thr Phe Arg Ser Glu Lys Gly Leu Leu Ser Ala 1760 1765 1770 Thr Gln Phe Thr Gln Pro Ala Leu Thr Leu Met Glu Lys Ala Ala 1775 1780 1785 Phe Glu Asp Leu Lys Ser Lys Gly Leu Ile Pro Ala Asp Ala Thr 1790 1795 1800 Phe Ala Gly His Ser Leu Gly Glu Tyr Ala Ala Leu Ala Ser Leu 1805 1810 1815 Ala Asp Val Met Ser Ile Glu Ser Leu Val Glu Val Val Phe Tyr 1820 1825 1830 Arg Gly Met Thr Met Gln Val Ala Val Pro Arg Asp Glu Leu Gly 1835 1840 1845 Arg Ser Asn Tyr Gly Met Ile Ala Ile Asn Pro Gly Arg Val Ala 1850 1855 1860 Ala Ser Phe Ser Gln Glu Ala Leu Gln Tyr Val Val Glu Arg Val 1865 1870 1875 Gly Lys Arg Thr Gly Trp Leu Val Glu Ile Val Asn Tyr Asn Val 1880 1885 1890 Glu Asn Gln Gln Tyr Val Ala Ala Gly Asp Leu Arg Ala Leu Asp 1895 1900 1905 Thr Val Thr Asn Val Leu Asn Phe Ile Lys Leu Gln Lys Ile Asp 1910 1915 1920 Ile Ile Glu Leu Gln Lys Ser Leu Ser Leu Glu Glu Val Glu Gly 1925 1930 1935 His Leu Phe Glu Ile Ile Asp Glu Ala Ser Lys Lys Ser Ala Val 1940 1945 1950 Lys Pro Arg Pro Leu Lys Leu Glu Arg Gly Phe Ala Cys Ile Pro 1955 1960 1965 Leu Val Gly Ile Ser Val Pro Phe His Ser Thr Tyr Leu Met Asn 1970 1975 1980 Gly Val Lys Pro Phe Lys Ser Phe Leu Lys Lys Asn Ile Ile Lys 1985 1990 1995 Glu Asn Val Lys Val Ala Arg Leu Ala Gly Lys Tyr Ile Pro Asn 2000 2005 2010 Leu Thr Ala Lys Pro Phe Gln Val Thr Lys Glu Tyr Phe Gln Asp 2015 2020 2025 Val Tyr Asp Leu Thr Gly Ser Glu Pro Ile Lys Glu Ile Ile Asp 2030 2035 2040 Asn Trp Glu Lys Tyr Glu Gln Ser 2045 2050 19 1887 PRT Saccharomyces cerevisiae 19 Met Lys Pro Glu Val Glu Gln Glu Leu Ala His Ile Leu Leu Thr Glu 1 5 10 15 Leu Leu Ala Tyr Gln Phe Ala Ser Pro Val Arg Trp Ile Glu Thr Gln 20 25 30 Asp Val Phe Leu Lys Asp Phe Asn Thr Glu Arg Val Val Glu Ile Gly 35 40 45 Pro Ser Pro Thr Leu Ala Gly Met Ala Gln Arg Thr Leu Lys Asn Lys 50 55 60 Tyr Glu Ser Tyr Asp Ala Ala Leu Ser Leu His Arg Glu Ile Leu Cys 65 70 75 80 Tyr Ser Lys Asp Ala Lys Glu Ile Tyr Tyr Thr Pro Asp Pro Ser Glu 85 90 95 Leu Ala Ala Lys Glu Glu Pro Ala Lys Glu Glu Ala Pro Ala Pro Thr 100 105 110 Pro Ala Ala Ser Ala Pro Ala Pro Ala Ala Ala Ala Pro Ala Pro Val 115 120 125 Ala Ala Ala Ala Pro Ala Ala Ala Ala Ala Glu Ile Ala Asp Glu Pro 130 135 140 Val Lys Ala Ser Leu Leu Leu His Val Leu Val Ala His Lys Leu Lys 145 150 155 160 Lys Ser Leu Asp Ser Ile Pro Met Ser Lys Thr Ile Lys Asp Leu Val 165 170 175 Gly Gly Lys Ser Thr Val Gln Asn Glu Ile Leu Gly Asp Leu Gly Lys 180 185 190 Glu Phe Gly Thr Thr Pro Glu Lys Pro Glu Glu Thr Pro Leu Glu Glu 195 200 205 Leu Ala Glu Thr Phe Gln Asp Thr Phe Ser Gly Ala Leu Gly Lys Gln 210 215 220 Ser Ser Ser Leu Leu Ser Arg Leu Ile Ser Ser Lys Met Pro Gly Gly 225 230 235 240 Phe Thr Ile Thr Val Ala Arg Lys Tyr Leu Gln Thr Arg Trp Gly Leu 245 250 255 Pro Ser Gly Arg Gln Asp Gly Val Leu Leu Val Ala Leu Ser Asn Glu 260 265 270 Pro Ala Ala Arg Leu Gly Ser Glu Ala Asp Ala Lys Ala Phe Leu Asp 275 280 285 Ser Met Ala Gln Lys Tyr Ala Ser Ile Val Gly Val Asp Leu Ser Ser 290 295 300 Ala Ala Ser Ala Ser Gly Ala Ala Gly Ala Gly Ala Ala Ala Gly Ala 305 310 315 320 Ala Met Ile Asp Ala Gly Ala Leu Glu Glu Ile Thr Lys Asp His Lys 325 330 335 Val Leu Ala Arg Gln Gln Leu Gln Val Leu Ala Arg Tyr Leu Lys Met 340 345 350 Asp Leu Asp Asn Gly Glu Arg Lys Phe Leu Lys Glu Lys Asp Thr Val 355 360 365 Ala Glu Leu Gln Ala Gln Leu Asp Tyr Leu Asn Ala Glu Leu Gly Glu 370 375 380 Phe Phe Val Asn Gly Val Ala Thr Ser Phe Ser Arg Lys Lys Ala Arg 385 390 395 400 Thr Phe Asp Ser Ser Trp Asn Trp Ala Lys Gln Ser Leu Leu Ser Leu 405 410 415 Tyr Phe Glu Ile Ile His Gly Val Leu Lys Asn Val Asp Arg Glu Val 420 425 430 Val Ser Glu Ala Ile Asn Ile Met Asn Arg Ser Asn Asp Ala Leu Ile 435 440 445 Lys Phe Met Glu Tyr His Ile Ser Asn Thr Asp Glu Thr Lys Gly Glu 450 455 460 Asn Tyr Gln Leu Val Lys Thr Leu Gly Glu Gln Leu Ile Glu Asn Cys 465 470 475 480 Lys Gln Val Leu Asp Val Asp Pro Val Tyr Lys Asp Val Ala Lys Pro 485 490 495 Thr Gly Pro Lys Thr Ala Ile Asp Lys Asn Gly Asn Ile Thr Tyr Ser 500 505 510 Glu Glu Pro Arg Glu Lys Val Arg Lys Leu Ser Gln Tyr Val Gln Glu 515 520 525 Met Ala Leu Gly Gly Pro Ile Thr Lys Glu Ser Gln Pro Thr Ile Glu 530 535 540 Glu Asp Leu Thr Arg Val Tyr Lys Ala Ile Ser Ala Gln Ala Asp Lys 545 550 555 560 Gln Asp Ile Ser Ser Ser Thr Arg Val Glu Phe Glu Lys Leu Tyr Ser 565 570 575 Asp Leu Met Lys Phe Leu Glu Ser Ser Lys Glu Ile Asp Pro Ser Gln 580 585 590 Thr Thr Gln Leu Ala Gly Met Asp Val Glu Asp Ala Leu Asp Lys Asp 595 600 605 Ser Thr Lys Glu Val Ala Ser Leu Pro Asn Lys Ser Thr Ile Ser Lys 610 615 620 Thr Val Ser Ser Thr Ile Pro Arg Glu Thr Ile Pro Phe Leu His Leu 625 630 635 640 Arg Lys Lys Thr Pro Ala Gly Asp Trp Lys Tyr Asp Arg Gln Leu Ser 645 650 655 Ser Leu Phe Leu Asp Gly Leu Glu Lys Ala Ala Phe Asn Gly Val Thr 660 665 670 Phe Lys Asp Lys Tyr Val Leu Ile Thr Gly Ala Gly Lys Gly Ser Ile 675 680 685 Gly Ala Glu Val Leu Gln Gly Leu Leu Gln Gly Gly Ala Lys Val Val 690 695 700 Val Thr Thr Ser Arg Phe Ser Lys Gln Val Thr Asp Tyr Tyr Gln Ser 705 710 715 720 Ile Tyr Ala Lys Tyr Gly Ala Lys Gly Ser Thr Leu Ile Val Val Pro 725 730 735 Phe Asn Gln Gly Ser Lys Gln Asp Val Glu Ala Leu Ile Glu Phe Ile 740 745 750 Tyr Asp Thr Glu Lys Asn Gly Gly Leu Gly Trp Asp Leu Asp Ala Ile 755 760 765 Ile Pro Phe Ala Ala Ile Pro Glu Gln Gly Ile Glu Leu Glu His Ile 770 775 780 Asp Ser Lys Ser Glu Phe Ala His Arg Ile Met Leu Thr Asn Ile Leu 785 790 795 800 Arg Met Met Gly Cys Val Lys Lys Gln Lys Ser Ala Arg Gly Ile Glu 805 810 815 Thr Arg Pro Ala Gln Val Ile Leu Pro Met Ser Pro Asn His Gly Thr 820 825 830 Phe Gly Gly Asp Gly Met Tyr Ser Glu Ser Lys Leu Ser Leu Glu Thr 835 840 845 Leu Phe Asn Arg Trp His Ser Glu Ser Trp Ala Asn Gln Leu Thr Val 850 855 860 Cys Gly Ala Ile Ile Gly Trp Thr Arg Gly Thr Gly Leu Met Ser Ala 865 870 875 880 Asn Asn Ile Ile Ala Glu Gly Ile Glu Lys Met Gly Val Arg Thr Phe 885 890 895 Ser Gln Lys Glu Met Ala Phe Asn Leu Leu Gly Leu Leu Thr Pro Glu 900 905 910 Val Val Glu Leu Cys Gln Lys Ser Pro Val Met Ala Asp Leu Asn Gly 915 920 925 Gly Leu Gln Phe Val Pro Glu Leu Lys Glu Phe Thr Ala Lys Leu Arg 930 935 940 Lys Glu Leu Val Glu Thr Ser Glu Val Arg Lys Ala Val Ser Ile Glu 945 950 955 960 Thr Ala Leu Glu His Lys Val Val Asn Gly Asn Ser Ala Asp Ala Ala 965 970 975 Tyr Ala Gln Val Glu Ile Gln Pro Arg Ala Asn Ile Gln Leu Asp Phe 980 985 990 Pro Glu Leu Lys Pro Tyr Lys Gln Val Lys Gln Ile Ala Pro Ala Glu 995 1000 1005 Leu Glu Gly Leu Leu Asp Leu Glu Arg Val Ile Val Val Thr Gly 1010 1015 1020 Phe Ala Glu Val Gly Pro Trp Gly Ser Ala Arg Thr Arg Trp Glu 1025 1030 1035 Met Glu Ala Phe Gly Glu Phe Ser Leu Glu Gly Cys Val Glu Met 1040 1045 1050 Ala Trp Ile Met Gly Phe Ile Ser Tyr His Asn Gly Asn Leu Lys 1055 1060 1065 Gly Arg Pro Tyr Thr Gly Trp Val Asp Ser Lys Thr Lys Glu Pro 1070 1075 1080 Val Asp Asp Lys Asp Val Lys Ala Lys Tyr Glu Thr Ser Ile Leu 1085 1090 1095 Glu His Ser Gly Ile Arg Leu Ile Glu Pro Glu Leu Phe Asn Gly 1100 1105 1110 Tyr Asn Pro Glu Lys Lys Glu Met Ile Gln Glu Val Ile Val Glu 1115 1120 1125 Glu Asp Leu Glu Pro Phe Glu Ala Ser Lys Glu Thr Ala Glu Gln 1130 1135 1140 Phe Lys His Gln His Gly Asp Lys Val Asp Ile Phe Glu Ile Pro 1145 1150 1155 Glu Thr Gly Glu Tyr Ser Val Lys Leu Leu Lys Gly Ala Thr Leu 1160 1165 1170 Tyr Ile Pro Lys Ala Leu Arg Phe Asp Arg Leu Val Ala Gly Gln 1175 1180 1185 Ile Pro Thr Gly Trp Asn Ala Lys Thr Tyr Gly Ile Ser Asp Asp 1190 1195 1200 Ile Ile Ser Gln Val Asp Pro Ile Thr Leu Phe Val Leu Val Ser 1205 1210 1215 Val Val Glu Ala Phe Ile Ala Ser Gly Ile Thr Asp Pro Tyr Glu 1220 1225 1230 Met Tyr Lys Tyr Val His Val Ser Glu Val Gly Asn Cys Ser Gly 1235 1240 1245 Ser Gly Met Gly Gly Val Ser Ala Leu Arg Gly Met Phe Lys Asp 1250 1255 1260 Arg Phe Lys Asp Glu Pro Val Gln Asn Asp Ile Leu Gln Glu Ser 1265 1270 1275 Phe Ile Asn Thr Met Ser Ala Trp Val Asn Met Leu Leu Ile Ser 1280 1285 1290 Ser Ser Gly Pro Ile Lys Thr Pro Val Gly Ala Cys Ala Thr Ser 1295 1300 1305 Val Glu Ser Val Asp Ile Gly Val Glu Thr Ile Leu Ser Gly Lys 1310 1315 1320 Ala Arg Ile Cys Ile Val Gly Gly Tyr Asp Asp Phe Gln Glu Glu 1325 1330 1335 Gly Ser Phe Glu Phe Gly Asn Met Lys Ala Thr Ser Asn Thr Leu 1340 1345 1350 Glu Glu Phe Glu His Gly Arg Thr Pro Ala Glu Met Ser Arg Pro 1355 1360 1365 Ala Thr Thr Thr Arg Asn Gly Phe Met Glu Ala Gln Gly Ala Gly 1370 1375 1380 Ile Gln Ile Ile Met Gln Ala Asp Leu Ala Leu Lys Met Gly Val 1385 1390 1395 Pro Ile Tyr Gly Ile Val Ala Met Ala Ala Thr Ala Thr Asp Lys 1400 1405 1410 Ile Gly Arg Ser Val Pro Ala Pro Gly Lys Gly Ile Leu Thr Thr 1415 1420 1425 Ala Arg Glu His His Ser Ser Val Lys Tyr Ala Ser Pro Asn Leu 1430 1435 1440 Asn Met Lys Tyr Arg Lys Arg Gln Leu Val Thr Arg Glu Ala Gln 1445 1450 1455 Ile Lys Asp Trp Val Glu Asn Glu Leu Glu Ala Leu Lys Leu Glu 1460 1465 1470 Ala Glu Glu Ile Pro Ser Glu Asp Gln Asn Glu Phe Leu Leu Glu 1475 1480 1485 Arg Thr Arg Glu Ile His Asn Glu Ala Glu Ser Gln Leu Arg Ala 1490 1495 1500 Ala Gln Gln Gln Trp Gly Asn Asp Phe Tyr Lys Arg Asp Pro Arg 1505 1510 1515 Ile Ala Pro Leu Arg Gly Ala Leu Ala Thr Tyr Gly Leu Thr Ile 1520 1525 1530 Asp Asp Leu Gly Val Ala Ser Phe His Gly Thr Ser Thr Lys Ala 1535 1540 1545 Asn Asp Lys Asn Glu Ser Ala Thr Ile Asn Glu Met Met Lys His 1550 1555 1560 Leu Gly Arg Ser Glu Gly Asn Pro Val Ile Gly Val Phe Gln Lys 1565 1570 1575 Phe Leu Thr Gly His Pro Lys Gly Ala Ala Gly Ala Trp Met Met 1580 1585 1590 Asn Gly Ala Leu Gln Ile Leu Asn Ser Gly Ile Ile Pro Gly Asn 1595 1600 1605 Arg Asn Ala Asp Asn Val Asp Lys Ile Leu Glu Gln Phe Glu Tyr 1610 1615 1620 Val Leu Tyr Pro Ser Lys Thr Leu Lys Thr Asp Gly Val Arg Ala 1625 1630 1635 Val Ser Ile Thr Ser Phe Gly Phe Gly Gln Lys Gly Gly Gln Ala 1640 1645 1650 Ile Val Val His Pro Asp Tyr Leu Tyr Gly Ala Ile Thr Glu Asp 1655 1660 1665 Arg Tyr Asn Glu Tyr Val Ala Lys Val Ser Ala Arg Glu Lys Ser 1670 1675 1680 Ala Tyr Lys Phe Phe His Asn Gly Met Ile Tyr Asn Lys Leu Phe 1685 1690 1695 Val Ser Lys Glu His Ala Pro Tyr Thr Asp Glu Leu Glu Glu Asp 1700 1705 1710 Val Tyr Leu Asp Pro Leu Ala Arg Val Ser Lys Asp Lys Lys Ser 1715 1720 1725 Gly Ser Leu Thr Phe Asn Ser Lys Asn Ile Gln Ser Lys Asp Ser 1730 1735 1740 Tyr Ile Asn Ala Asn Thr Ile Glu Thr Ala Lys Met Ile Glu Asn 1745 1750 1755 Met Thr Lys Glu Lys Val Ser Asn Gly Gly Val Gly Val Asp Val 1760 1765 1770 Glu Leu Ile Thr Ser Ile Asn Val Glu Asn Asp Thr Phe Ile Glu 1775 1780 1785 Arg Asn Phe Thr Pro Gln Glu Ile Glu Tyr Cys Ser Ala Gln Pro 1790 1795 1800 Ser Val Gln Ser Ser Phe Ala Gly Thr Trp Ser Ala Lys Glu Ala 1805 1810 1815 Val Phe Lys Ser Leu Gly Val Lys Ser Leu Gly Gly Gly Ala Ala 1820 1825 1830 Leu Lys Asp Ile Glu Ile Val Arg Val Asn Lys Asn Ala Pro Ala 1835 1840 1845 Val Glu Leu His Gly Asn Ala Lys Lys Ala Ala Glu Glu Ala Gly 1850 1855 1860 Val Thr Asp Val Lys Val Ser Ile Ser His Asp Asp Leu Gln Ala 1865 1870 1875 Val Ala Val Ala Val Ser Thr Lys Lys 1880 1885 20 2037 PRT Candida albicans 20 Met Ser Thr His Arg Pro Phe Gln Leu Thr His Gly Ser Ile Glu His 1 5 10 15 Thr Leu Leu Val Pro Asn Asp Leu Phe Phe Asn Tyr Ser Gln Leu Lys 20 25 30 Asp Glu Phe Ile Lys Thr Leu Pro Glu Pro Thr Glu Gly Phe Ala Gly 35 40 45 Asp Asp Glu Pro Ser Ser Pro Ala Glu Leu Tyr Gly Lys Phe Ile Gly 50 55 60 Phe Ile Ser Asn Ala Gln Phe Pro Gln Ile Val Glu Leu Ser Leu Lys 65 70 75 80 Asp Phe Glu Ser Arg Phe Leu Asp Asn Asn Asn Asp Asn Ile His Ser 85 90 95 Phe Ala Val Lys Leu Leu Asp Asp Glu Thr Tyr Pro Thr Thr Ile Ala 100 105 110 Lys Val Lys Glu Asn Ile Val Lys Asn Tyr Tyr Lys Ala Val Lys Ser 115 120 125 Ile Asn Lys Val Glu Ser Asn Leu Leu Tyr His Cys Lys His Asp Ala 130 135 140 Lys Leu Val Ala Ile Phe Gly Gly Gln Gly Asn Thr Asp Asp Tyr Phe 145 150 155 160 Glu Glu Leu Arg Glu Leu Tyr Thr Leu Tyr Gln Gly Leu Ile Glu Asp 165 170 175 Leu Leu Val Ser Ile Ala Glu Lys Leu Asn Gln Leu His Pro Ser Phe 180 185 190 Asp Lys Ile Tyr Thr Gln Gly Leu Asn Ile Leu Ser Trp Leu Lys His 195 200 205 Pro Glu Thr Thr Pro Asp Gln Asp Tyr Leu Leu Ser Val Pro Val Ser 210 215 220 Cys Pro Val Ile Cys Val Ile Gln Leu Cys His Tyr Thr Ile Thr Cys 225 230 235 240 Lys Val Leu Gly Leu Thr Pro Gly Glu Phe Arg Asn Ser Leu Lys Trp 245 250 255 Ser Thr Gly His Ser Gln Gly Leu Val Thr Ala Val Thr Ile Ala Ala 260 265 270 Ser Asp Ser Trp Asp Ser Phe Leu Lys Asn Ser Leu Thr Ala Val Ser 275 280 285 Leu Leu Leu Phe Ile Gly Ser Arg Cys Leu Ser Thr Tyr Pro Arg Thr 290 295 300 Ser Leu Pro Pro Thr Met Leu Gln Asp Ser Leu Asp Asn Gly Glu Gly 305 310 315 320 Arg Pro Ser Pro Met Leu Ser Val Arg Asp Leu Ser Ile Lys Gln Val 325 330 335 Glu Lys Phe Ile Glu Gln Thr Asn Ser His Leu Pro Arg Glu Lys His 340 345 350 Ile Ala Ile Ser Leu Ile Asn Gly Ala Arg Asn Leu Val Leu Ser Gly 355 360 365 Pro Pro Glu Ser Leu Tyr Gly Phe Asn Leu Asn Leu Arg Asn Gln Lys 370 375 380 Ala Pro Met Gly Leu Asp Gln Ser Arg Val Pro Phe Ser Glu Arg Lys 385 390 395 400 Leu Lys Cys Ser Asn Arg Phe Leu Pro Ile Phe Ala Pro Phe His Ser 405 410 415 His Leu Leu Ala Asp Ala Thr Glu Leu Ile Leu Asp Asp Val Lys Glu 420 425 430 His Gly Leu Ser Phe Glu Gly Leu Lys Ile Pro Val Tyr Asp Thr Phe 435 440 445 Asp Gly Ser Asp Phe Gln Ala Leu Lys Glu Pro Ile Ile Asp Arg Val 450 455 460 Val Lys Leu Ile Thr Glu Leu Pro Val His Trp Glu Glu Ala Thr Asn 465 470 475 480 His Lys Ala Thr His Ile Leu Asp Phe Gly Pro Gly Gly Val Ser Gly 485 490 495 Leu Gly Val Leu Thr His Arg Asn Lys Glu Gly Thr Gly Ala Arg Ile 500 505 510 Ile Leu Ala Gly Thr Leu Asp Ser Asn Pro Ile Asp Asp Glu Tyr Gly 515 520 525 Phe Lys His Glu Ile Phe Gln Thr Ser Ala Asp Lys Ala Ile Lys Trp 530 535 540 Ala Pro Asp Trp Leu Lys Glu Leu Arg Pro Thr Leu Val Lys Asn Ser 545 550 555 560 Glu Gly Lys Ile Tyr Val Lys Thr Lys Phe Ser Gln Leu Leu Gly Arg 565 570 575 Ala Pro Leu Met Val Ala Gly Met Thr Pro Thr Thr Val Asn Thr Asp 580 585 590 Ile Val Ser Ala Ser Leu Asn Ala Gly Tyr His Ile Glu Leu Ala Gly 595 600 605 Gly Gly Tyr Phe Ser Pro Val Met Met Thr Arg Ala Ile Asp Asp Ile 610 615 620 Val Ser Arg Ile Lys Pro Gly Tyr Gly Leu Gly Ile Asn Leu Ile Tyr 625 630 635 640 Val Asn Pro Phe Met Leu Gln Trp Gly Ile Pro Leu Ile Lys Asp Leu 645 650 655 Arg Glu Lys Gly Tyr Pro Ile Gln Ser Leu Thr Ile Gly Ala Gly Val 660 665 670 Pro Ser Ile Glu Val Ala Thr Glu Tyr Ile Glu Asp Leu Gly Leu Thr 675 680 685 His Leu Gly Leu Lys Pro Gly Ser Val Asp Ala Ile Ser Gln Val Ile 690 695 700 Ala Ile Ala Lys Ala His Pro Thr Phe Pro Ile Val Leu Gln Trp Thr 705 710 715 720 Gly Gly Arg Gly Gly Gly His His Ser Phe Glu Asp Phe His Gln Pro 725 730 735 Ile Ile Gln Met Tyr Ser Lys Ile Arg Arg Cys Ser Asn Ile Val Leu 740 745 750 Val Ala Gly Ser Gly Phe Gly Ser Asp Glu Asp Thr Tyr Pro Tyr Leu 755 760 765 Ser Gly Tyr Trp Ser Glu Lys Phe Asn Tyr Pro Pro Met Pro Phe Asp 770 775 780 Gly Val Leu Phe Gly Ser Arg Val Met Thr Ser Lys Glu Ser His Thr 785 790 795 800 Ser Leu Ala Ala Lys Lys Leu Ile Val Glu Cys Lys Gly Val Pro Asp 805 810 815 Gln Gln Trp Glu Gln Thr Tyr Lys Lys Pro Thr Gly Gly Ile Ile Thr 820 825 830 Val Arg Ser Glu Met Gly Glu Pro Ile His Lys Ile Ala Thr Arg Gly 835 840 845 Val Met Phe Trp Lys Glu Leu Asp Asp Thr Ile Phe Asn Leu Pro Lys 850 855 860 Asn Lys Leu Leu Asp Ala Leu Asn Lys Lys Arg Asp His Ile Ile Lys 865 870 875 880 Lys Leu Asn Asn Asp Phe Gln Lys Pro Trp Phe Gly Lys Asn Ala Asn 885 890 895 Gly Val Cys Asp Leu Gln Glu Met Thr Tyr Lys Glu Val Ala Asn Arg 900 905 910 Leu Val Glu Leu Met Tyr Val Lys Lys Ser His Arg Trp Ile Asp Val 915 920 925 Ser Leu Arg Asn Met Tyr Gly Asp Phe Leu Arg Arg Val Glu Glu Arg 930 935 940 Phe Thr Ser Ser Ala Gly Thr Val Ser Leu Leu Gln Asn Phe Asn Gln 945 950 955 960 Leu Asn Glu Pro Glu Gln Phe Thr Ala Asp Phe Phe Glu Lys Phe Pro 965 970 975 Gln Ala Gly Lys Gln Leu Ile Ser Glu Glu Asp Cys Asp Tyr Phe Leu 980 985 990 Met Leu Ala Ala Arg Pro Gly Gln Lys Pro Val Pro Phe Val Pro Val 995 1000 1005 Leu Asp Glu Arg Phe Glu Phe Phe Phe Lys Lys Asp Ser Leu Trp 1010 1015 1020 Gln Ser Glu Asp Leu Glu Ser Val Val Asp Glu Asp Val Gln Arg 1025 1030 1035 Thr Cys Ile Leu His Gly Pro Val Ala Ser Gln Tyr Thr Ser Lys 1040 1045 1050 Val Asp Glu Pro Ile Gly Asp Ile Leu Asn Ser Ile His Glu Gly 1055 1060 1065 His Ile Ala Arg Leu Ile Lys Glu Glu Tyr Ala Gly Asp Glu Ser 1070 1075 1080 Lys Ile Pro Val Val Glu Tyr Phe Gly Gly Lys Lys Pro Ala Ser 1085 1090 1095 Val Ser Ala Thr Ser Val Asn Ile Ile Asp Gly Asn Gln Val Val 1100 1105 1110 Tyr Glu Ile Asp Ser Glu Leu Pro Asn Lys Gln Glu Trp Leu Asp 1115 1120 1125 Leu Leu Ala Gly Thr Glu Leu Asn Trp Leu Gln Ala Phe Ile Ser 1130 1135 1140 Thr Asp Arg Ile Val Gln Gly Ser Lys His Val Ser Asn Pro Leu 1145 1150 1155 His Asp Ile Leu Thr Pro Ala Lys His Ser Lys Val Thr Ile Asp 1160 1165 1170 Lys Lys Thr Lys Lys Leu Thr Ala Phe Glu Asn Ile Lys Gly Asp 1175 1180 1185 Leu Leu Pro Val Val Glu Ile Glu Leu Val Lys Pro Asn Thr Ile 1190 1195 1200 Gln Leu Ser Leu Ile Glu His Arg Thr Ala Asp Thr Asn Pro Val 1205 1210 1215 Ala Leu Pro Phe Leu Tyr Lys Tyr Asn Pro Ala Asp Gly Phe Ala 1220 1225 1230 Pro Ile Leu Glu Ile Met Glu Asp Arg Asn Glu Arg Ile Lys Glu 1235 1240 1245 Phe Tyr Trp Lys Leu Trp Phe Gly Ser Ser Val Pro Tyr Ser Asn 1250 1255 1260 Asp Ile Asn Val Glu Lys Ala Ile Leu Gly Asp Glu Ile Thr Ile 1265 1270 1275 Ser Ser Gln Thr Ile Ser Glu Phe Thr His Ala Ile Gly Asn Lys 1280 1285 1290 Cys Asp Ala Phe Val Asp Arg Pro Gly Lys Ala Thr Leu Ala Pro 1295 1300 1305 Met Asp Phe Ala Ile Val Ile Gly Trp Lys Ala Ile Ile Lys Ala 1310 1315 1320 Ile Phe Pro Lys Ser Val Asp Gly Asp Leu Leu Lys Leu Val His 1325 1330 1335 Leu Ser Asn Gly Tyr Lys Met Ile Thr Gly Ala Ala Pro Leu Lys 1340 1345 1350 Lys Gly Asp Val Val Ser Thr Lys Ala Glu Ile Lys Ala Val Leu 1355 1360 1365 Asn Gln Pro Ser Gly Lys Leu Val Glu Val Val Gly Thr Ile Tyr 1370 1375 1380 Arg Glu Gly Lys Pro Val Met Glu Val Thr Ser Gln Phe Leu Tyr 1385 1390 1395 Arg Gly Glu Tyr Asn Asp Tyr Cys Asn Thr Phe Gln Lys Val Thr 1400 1405 1410 Glu Thr Pro Val Gln Val Ala Phe Lys Ser Ala Lys Asp Leu Ala 1415 1420 1425 Val Leu Arg Ser Lys Glu Trp Phe His Leu Glu Lys Asp Val Gln 1430 1435 1440 Phe Asp Val Leu Thr Phe Arg Cys Glu Ser Thr Tyr Lys Phe Lys 1445 1450 1455 Ser Ala Asn Val Tyr Ser Ser Ile Lys Thr Thr Gly Gln Val Leu 1460 1465 1470 Leu Glu Leu Pro Thr Lys Glu Val Ile Gln Val Gly Ser Val Asp 1475 1480 1485 Tyr Glu Ala Gly Thr Ser Tyr Gly Asn Pro Val Thr Asp Tyr Leu 1490 1495 1500 Ser Arg Asn Gly Lys Thr Ile Glu Glu Ser Val Ile Phe Glu Asn 1505 1510 1515 Ala Ile Pro Leu Ser Ser Gly Glu Glu Leu Thr Ser Lys Ala Pro 1520 1525 1530 Gly Thr Asn Glu Pro Tyr Ala Ile Val Ser Gly Asp Tyr Asn Pro 1535 1540 1545 Ile His Val Ser Arg Val Phe Ala Ala Tyr Ala Lys Leu Pro Gly 1550 1555 1560 Thr Ile Thr His Gly Met Tyr Ser Ser Ala Ser Ile Arg Ala Leu 1565 1570 1575 Val Glu Glu Trp Ala Ala Asn Asn Val Ala Ala Arg Val Arg Ala 1580 1585 1590 Phe Lys Cys Asp Phe Val Gly Met Val Leu Pro Asn Asp Thr Leu 1595 1600 1605 Gln Thr Thr Met Glu His Val Gly Met Ile Asn Gly Arg Lys Ile 1610 1615 1620 Ile Lys Val Glu Thr Arg Asn Val Glu Thr Glu Leu Pro Val Leu 1625 1630 1635 Ile Gly Glu Ala Glu Ile Glu Gln Pro Thr Thr Thr Tyr Val Phe 1640 1645 1650 Thr Gly Gln Gly Ser Gln Glu Gln Gly Met Gly Met Glu Leu Tyr 1655 1660 1665 Asn Ser Ser Glu Val Ala Arg Glu Val Trp Asp Lys Ala Asp Arg 1670 1675 1680 His Phe Val Asn Asn Tyr Gly Phe Ser Ile Leu Asp Ile Val Gln 1685 1690 1695 Asn Asn Pro Asn Glu Leu Thr Ile His Phe Gly Gly Ala Lys Gly 1700 1705 1710 Arg Ala Ile Arg Asp Asn Tyr Ile Gly Met Met Phe Glu Thr Ile 1715 1720 1725 Gly Glu Asp Gly Ala Leu Lys Ser Glu Lys Ile Phe Lys Asp Ile 1730 1735 1740 Asp Glu Thr Thr Thr Ser Tyr Thr Phe Val Ser Pro Thr Gly Leu 1745 1750 1755 Leu Ser Ala Thr Gln Phe Thr Gln Pro Ala Leu Thr Leu Met Glu 1760 1765 1770 Lys Ala Ala Tyr Glu Asp Ile Lys Ser Lys Gly Leu Ile Pro Ser 1775 1780 1785 Asp Ile Met Phe Ala Gly His Ser Leu Gly Glu Tyr Ser Ala Leu 1790 1795 1800 Ser Ser Leu Ala Asn Val Met Pro Ile Glu Ser Leu Val Asp Val 1805 1810 1815 Val Phe Tyr Arg Gly Met Thr Met Gln Val Ala Val Pro Arg Asp 1820 1825 1830 Glu Leu Gly Arg Ser Asn Tyr Gly Met Val Ala Val Asn Pro Ser 1835 1840 1845 Arg Val Ser Ala Thr Phe Asp Asp Ser Ala Leu Arg Phe Val Val 1850 1855 1860 Asp Glu Val Ala Asn Lys Thr Lys Trp Leu Leu Glu Ile Val Asn 1865 1870 1875 Tyr Asn Val Glu Asn Gln Gln Tyr Val Ala Ala Gly Asp Leu Arg 1880 1885 1890 Ala Leu Asp Thr Leu Thr Asn Val Leu Asn Val Leu Lys Ile Asn 1895 1900 1905 Lys Ile Asp Ile Val Lys Leu Gln Glu Gln Met Ser Ile Glu Lys 1910 1915 1920 Val Lys Glu His Leu Tyr Glu Ile Val Asp Glu Val Ala Ala Lys 1925 1930 1935 Ser Leu Ala Lys Pro Gln Pro Ile Asp Leu Glu Arg Gly Phe Ala 1940 1945 1950 Val Ile Pro Leu Lys Gly Ile Ser Val Pro Phe His Ser Ser Tyr 1955 1960 1965 Leu Met Ser Gly Val Lys Pro Phe Gln Arg Phe Leu Cys Lys Lys 1970 1975 1980 Ile Pro Lys Ser Ser Val Lys Pro Gln Asp Leu Ile Gly Lys Tyr 1985 1990 1995 Ile Pro Asn Leu Thr Ala Lys Pro Phe Glu Leu Thr Lys Glu Tyr 2000 2005 2010 Phe Gln Ser Val Tyr Asp Leu Thr Lys Ser Glu Lys Ile Lys Ser 2015 2020 2025 Ile Leu Asp Asn Trp Glu Gln Tyr Glu 2030 2035 21 6072 DNA Candida albicans 21 ggatcctttt ttttttgggt aatattaaca atccagctta ggccatattg ttgggtgtcc 60 ttaaaaatta tgtgccaatt atttacttat atattgatat agctctcctt ttctcttttt 120 tatatttttc aaagtttttt ttattctttt actgtttatt caactaactt gtttttattt 180 ctcccccaat taacaatgaa accagaaatt gaacaagaat tatcccacac tttgttaact 240 gaattgttgg catatcaatt tgcttctcca gttagatgga ttgaaactca agatgtcttt 300 ttaaaacagc ataatactga aagaatcatc gaaattggtc cttcaccaac tttagctggt 360 atggccaata gaactatcaa agccaaatat gaatcctatg atgctgcttt atctttgcaa 420 cgacaagtct tgtgttactc caaagatgct aaggagattt actacaagcc agatccagca 480 gatcttgctc ctaaggaaac accaaagcaa gaagagagta ccccatcagc tcctgccgct 540 gccactccaa cacctgctgc tgccgctgct cctactccag caccagctcc tgcaagtgct 600 ggcccagttg aatctattcc agatgaacca gtcaaggcta acttgttaat ccatgttttg 660 gttgcacaaa aattaaagaa acctttagat gctgttccaa tgaccaaggc aattaaggat 720 ttggttaatg gtaaatccac tgttcaaaat gaaattcttg gtgacttggg taaggaattt 780 ggctctactc ctgaaaaacc ggaagacact ccattggaag aattagctga acaattccaa 840 gattcattca gcggtcaatt aggaaagact tctacttcat tgattggtag attaatgtcc 900 tcaaagatgc cgggtggatt ttccatcact actgctagaa agtatttgga atcaagattt 960 ggtttgggtg ctggtagaca agattctgtc ttgttgatgg ctttaacaaa tgaaccagct 1020 aatagattag gttctgaagc cgatgcaaaa actttctttg atggaattgc tcaaaaatac 1080 gcatcaagtg ctgggatctc cttgtcatca ggagcaggct ccggtgcagg cgccgcaaat 1140 agtggtggtg ctgttgttga tagtgctgcc ttagatgctt taacagctga aaacaagaaa 1200 ttagccaaac agcaattaga agttttagca agatacttgc aaagtcgact taaacaaggg 1260 agccttaaat cttttatcaa ggaaaaggaa gcttctgctg ttttacaaaa agagttagat 1320 ttgtgggaag cagaacacgg agaattctat gctaagggta tccaaccaac tttctccgca 1380 ttaaagtcta gaacttatga ctcctattgg aattgggccc gtcaagacgt tttatcaatg 1440 tatttcgaca ttatttttgg caagttaact tctgttgata gagaaaccat caaccaatgt 1500 attcaaatca tgaacagagc caatccaact ttaatcaagt ttatgcaata tcatatcgac 1560 cattgtccag aatataaagg tgaaacttat aaattggcca agagattggg tcaacaattg 1620 attgacaact gtaaacaagt tttgactgaa gatccagttt acaaagatgt ttccagaatt 1680 actggtccaa agactaaagt cagtgctaag ggtaacattg aatatgagga aactcaaaag 1740 gattcagtta gaaaatttga acaatatgtg tatgaaatgg cccaaggtgg tgctatgacc 1800 aaagttagtc aaccaactat tcaagaagat ttagctagag tttacaaggc tatttccaaa 1860 caagcttcca aagatagcaa attggaattg caaagagttt acgaagattt attgaaggtg 1920 gttgaaagtt ccaaggaaat cgaaaccgaa caattgacta aagatatttt acaagctgct 1980 acagttccaa caaccccaac agaggaagta gacgatcctt gtactccttc ttcggatgat 2040 gaaattgctt ctttaccaga taagacttct atcattcaac ctgtctcgtc tactattcca 2100 tctcaaacta ttccattttt gcacattcag aaaaagacca aagacggttg ggaatacaat 2160 aagaaattat cttctcttta cttggatgga ttggaatcag ctgccattaa tggtttaact 2220 ttcaaagaca agtatgtctt agttactggt gctggtgctg gctctattgg tgccgaaatt 2280 ttgcaaggtt taatcagtgg tggtgccaaa gttattgtca caacctctag attttccaag 2340 aaagttaccg agtattatca aaacatgtat gccagatatg gtgctgctgg gtctacttta 2400 attgttgttc cgttcaacca aggttctaaa caagatgttg atgcattggt tcaatacatt 2460 tatgatgagc caaagaaagg tggtttgggt tgggatttgg atgcaatcat tccatttgct 2520 gctattccag aaaatggtaa tggtctcgac aacattgatt ctaaatctga atttgcccac 2580 agaatcatgt tgaccaacct tttaagattg ttaggtgctg ttaaatccaa aaagcccact 2640 gacactagac ctgctcaatg tattttgcca ttatctccaa atcacggaac ttttggtttt 2700 gacgggttgt actctgaatc taaaatctca ttggaaacct tattcaacag atggtattct 2760 gaagattggg gatccaagtt gactgtttgt ggtgccgtaa ttgggtggac tagaggtaca 2820 ggtttgatga gtgccaataa cattattgct gaaggtattg aaaaattggg tgtcagaact 2880 ttctcccaaa aggaaatggc tttcaatatt ttaggtttat tgacaccaga aattgtacaa 2940 ttatgtcaag aagaaccagt tatggctgac ttgaatggtg gtttgcaatt cattgacaac 3000 ttgaaggatt tcacatctaa attaagaacc gacttgttgg aaactgcaga cattagaaga 3060 gctgtttcta ttgaatcagc tatcgagcaa aaagttgtca atggtgacaa tgtcgatgca 3120 aactactcaa aggttatggt tgaacctaga gccaacatga aatttgattt cccaactttg 3180 aaatcttatg atgaaatcaa acaaattgct ccagaattgg aaggtatgtt ggatttggaa 3240 aatgttgtcg ttgtgacagg ttttgctgaa gttggtccat ggggtaactc tagaaccaga 3300 tgggaaatgg aagcttatgg tgagttctca ttggaaggtg ccattgaaat ggcttggatt 3360 atgggtttca tcaagtatca taatggtaat ttgcaaggga aaccatactc tggatgggtt 3420 gatgccaaga ctcaaactcc aattgacgaa aaggatatca aatccaaata tgaagaagaa 3480 attttagaac attccggtat tagattgatt gagccagaat tgttcaatgg ctatgatcca 3540 aagaaaaaac aaatgattca agaaattgtt gttcaacacg atttagaacc atttgaatgt 3600 tctaaagaaa cagctgagca atacaaacac gaacacggag aaaaatgtga aatttttgaa 3660 attgaagaaa gtggtgaata cacagttaga atcttgaaag gtgcaacatt gtacgttccg 3720 aaagctttga gatttgatag attagttgct ggtcaaattc caactggttg ggacgctcgt 3780 acctatggta tcccagaaga cactattagt caagttgatc caatcacttt gtacgtgttg 3840 gttgccactg ttgaagcctt gttgtctgct ggtattactg atccatatga attctacaaa 3900 tacgttcatg tgtctgaagt tggtaactgt tctggttccg gtatgggagg tgtctctgct 3960 ttgagaggaa tgttcaaaga tagatatgct gacaaaccag ttcaaaatga cattttgcaa 4020 gaatcattta tcaacactat gtctgcttgg gtcaatatgt tgttgttgtc ttcctctggt 4080 ccaatcaaga caccagtcgg tgcttgtgcc actgctgttg aatcggttga cattggtatt 4140 gaaacaattt tgtctggtaa agctaaagta gttttggtag gtggttacga tgacttccaa 4200 gaagaagggt cttatgaatt cgccaatatg aatgctactt ctaattctat tgaagagttc 4260 aaacacggaa gaacaccaaa ggaaatgtca agaccaacta ctactaccag aaatggtttc 4320 atggaagctc aaggttctgg tattcaagtt atcatgactg ctgatttggc tctcaagatg 4380 ggtgttccaa tccacgctgt attggccatg actgctactg ccactgataa gattggtaga 4440 tctgttccag caccaggtaa aggtattttg accactgcca gagaacatca tggcaacttg 4500 aagtacccat ctccactttt gaacatcaag tacaggaaga gacaattgaa caaaagattg 4560 gaacaaatca aatcttggga agaaacagaa ctttcttact tgcaagaaga agccgagttg 4620 gccaaagaag aatttggtga cgaattttct atgcatgagt tcttgaaaga gagaactgaa 4680 gaagtgtacc gtgaatcaaa gagacaagtt tctgatgcta agaaacaatg gggtaattca 4740 ttctacaagt ctgatccaag aattgctcca ttgagaggag cattggctgc cttcaactta 4800 accatcgatg atattggtgt tgcatccttc catggtactt ccaccgttgc taacgataag 4860 aatgaatctg ccacaatcaa caatatgatg aaacacttgg gtagatccga aggtaaccca 4920 gtatttggtg ttttccaaaa atacttgaca ggtcatccaa aaggtgcagc tggtgcttgg 4980 atgttgaatg gtgccattca aattcttgag tctggtcttg ttccaggtaa cagaaatgcg 5040 gataatgttg ataagctttt agaacaatac gaatatgtat tgtacccatc aagatcaatt 5100 caaaccgatg gtattaaagc cgtttctgtt acatcatttg gtttcggtca aaaaggtgca 5160 caagccgttg ttgttcatcc agattactta tttgctgttt tggatagatc cacttatgaa 5220 gaatatgcta ctaaggtctc tgctagaaat aaaaagacct accgttacat gcacaatgca 5280 atcaccagaa acactatgtt tgttgccaaa gacaaagctc catatagtga cgaattggaa 5340 caaccagttt acttggatcc attggctcgt gttgaagaaa acaagaaaaa gttggtattc 5400 agtgacaaaa caattcaatc gaaccaatct tatgttggag aagttgctca aaaaactgct 5460 aaggcattgt ctactttaaa caaatcatca aagggagttg gtgtagatgt tgaattgttg 5520 tcagcaatca atatcgacaa tgaaaccttt attgaaagaa actttactgg taatgaagtt 5580 gaatactgtt tgaatactgc tcacccacaa gcttcattca ctggaacttg gtcagcaaag 5640 gaagctgttt tcaaagcctt gggtgttgaa tcaaaaggtg ctggagcaag cttgattgat 5700 attgaaatca ctcgtgacgt taatggtgct cctaaagtaa ttttgcatgg tgaggccaaa 5760 aaagctgctg ctaaagctgg tgttaaaaat gtcaatattt caatttctca tgatgatttc 5820 caagctactg ctgttgcttt aagtgaattt taaaattagt agtgtttaga aatattcgtg 5880 tatatctgat caaaaacttt tttgattttt aatatatgtc cggttgtaca attttttttt 5940 ctgttgattt aaactgatct cattattttg ttctctcaca gctcacagcc tacaaccata 6000 aaaaaagccc aacactcact tttgctcact ggttcaccac cactacggaa aaaataagaa 6060 caacaaataa aa 6072 22 1885 PRT Candida albicans 22 Met Lys Pro Glu Ile Glu Gln Glu Leu Ser His Thr Leu Leu Thr Glu 1 5 10 15 Leu Leu Ala Tyr Gln Phe Ala Ser Pro Val Arg Trp Ile Glu Thr Gln 20 25 30 Asp Val Phe Leu Lys Gln His Asn Thr Glu Arg Ile Ile Glu Ile Gly 35 40 45 Pro Ser Pro Thr Leu Ala Gly Met Ala Asn Arg Thr Ile Lys Ala Lys 50 55 60 Tyr Glu Ser Tyr Asp Ala Ala Leu Ser Leu Gln Arg Gln Val Leu Cys 65 70 75 80 Tyr Ser Lys Asp Ala Lys Glu Ile Tyr Tyr Lys Pro Asp Pro Ala Asp 85 90 95 Leu Ala Pro Lys Glu Thr Pro Lys Gln Glu Glu Ser Thr Pro Ser Ala 100 105 110 Pro Ala Ala Ala Thr Pro Thr Pro Ala Ala Ala Ala Ala Pro Thr Pro 115 120 125 Ala Pro Ala Pro Ala Ser Ala Gly Pro Val Glu Ser Ile Pro Asp Glu 130 135 140 Pro Val Lys Ala Asn Leu Leu Ile His Val Leu Val Ala Gln Lys Leu 145 150 155 160 Lys Lys Pro Leu Asp Ala Val Pro Met Thr Lys Ala Ile Lys Asp Leu 165 170 175 Val Asn Gly Lys Ser Thr Val Gln Asn Glu Ile Leu Gly Asp Leu Gly 180 185 190 Lys Glu Phe Gly Ser Thr Pro Glu Lys Pro Glu Asp Thr Pro Leu Glu 195 200 205 Glu Leu Ala Glu Gln Phe Gln Asp Ser Phe Ser Gly Gln Leu Gly Lys 210 215 220 Thr Ser Thr Ser Leu Ile Gly Arg Leu Met Ser Ser Lys Met Pro Gly 225 230 235 240 Gly Phe Ser Ile Thr Thr Ala Arg Lys Tyr Leu Glu Ser Arg Phe Gly 245 250 255 Leu Gly Ala Gly Arg Gln Asp Ser Val Leu Leu Met Ala Leu Thr Asn 260 265 270 Glu Pro Ala Asn Arg Leu Gly Ser Glu Ala Asp Ala Lys Thr Phe Phe 275 280 285 Asp Gly Ile Ala Gln Lys Tyr Ala Ser Ser Ala Gly Ile Ser Leu Ser 290 295 300 Ser Gly Ala Gly Ser Gly Ala Gly Ala Ala Asn Ser Gly Gly Ala Val 305 310 315 320 Val Asp Ser Ala Ala Leu Asp Ala Leu Thr Ala Glu Asn Lys Lys Leu 325 330 335 Ala Lys Gln Gln Leu Glu Val Leu Ala Arg Tyr Leu Gln Ser Arg Leu 340 345 350 Lys Gln Gly Ser Leu Lys Ser Phe Ile Lys Glu Lys Glu Ala Ser Ala 355 360 365 Val Leu Gln Lys Glu Leu Asp Leu Trp Glu Ala Glu His Gly Glu Phe 370 375 380 Tyr Ala Lys Gly Ile Gln Pro Thr Phe Ser Ala Leu Lys Ser Arg Thr 385 390 395 400 Tyr Asp Ser Tyr Trp Asn Trp Ala Arg Gln Asp Val Leu Ser Met Tyr 405 410 415 Phe Asp Ile Ile Phe Gly Lys Leu Thr Ser Val Asp Arg Glu Thr Ile 420 425 430 Asn Gln Cys Ile Gln Ile Met Asn Arg Ala Asn Pro Thr Leu Ile Lys 435 440 445 Phe Met Gln Tyr His Ile Asp His Cys Pro Glu Tyr Lys Gly Glu Thr 450 455 460 Tyr Lys Leu Ala Lys Arg Leu Gly Gln Gln Leu Ile Asp Asn Cys Lys 465 470 475 480 Gln Val Leu Thr Glu Asp Pro Val Tyr Lys Asp Val Ser Arg Ile Thr 485 490 495 Gly Pro Lys Thr Lys Val Ser Ala Lys Gly Asn Ile Glu Tyr Glu Glu 500 505 510 Thr Gln Lys Asp Ser Val Arg Lys Phe Glu Gln Tyr Val Tyr Glu Met 515 520 525 Ala Gln Gly Gly Ala Met Thr Lys Val Ser Gln Pro Thr Ile Gln Glu 530 535 540 Asp Leu Ala Arg Val Tyr Lys Ala Ile Ser Lys Gln Ala Ser Lys Asp 545 550 555 560 Ser Lys Leu Glu Leu Gln Arg Val Tyr Glu Asp Leu Leu Lys Val Val 565 570 575 Glu Ser Ser Lys Glu Ile Glu Thr Glu Gln Leu Thr Lys Asp Ile Leu 580 585 590 Gln Ala Ala Thr Val Pro Thr Thr Pro Thr Glu Glu Val Asp Asp Pro 595 600 605 Cys Thr Pro Ser Ser Asp Asp Glu Ile Ala Ser Leu Pro Asp Lys Thr 610 615 620 Ser Ile Ile Gln Pro Val Ser Ser Thr Ile Pro Ser Gln Thr Ile Pro 625 630 635 640 Phe Leu His Ile Gln Lys Lys Thr Lys Asp Gly Trp Glu Tyr Asn Lys 645 650 655 Lys Leu Ser Ser Leu Tyr Leu Asp Gly Leu Glu Ser Ala Ala Ile Asn 660 665 670 Gly Leu Thr Phe Lys Asp Lys Tyr Val Leu Val Thr Gly Ala Gly Ala 675 680 685 Gly Ser Ile Gly Ala Glu Ile Leu Gln Gly Leu Ile Ser Gly Gly Ala 690 695 700 Lys Val Ile Val Thr Thr Ser Arg Phe Ser Lys Lys Val Thr Glu Tyr 705 710 715 720 Tyr Gln Asn Met Tyr Ala Arg Tyr Gly Ala Ala Gly Ser Thr Leu Ile 725 730 735 Val Val Pro Phe Asn Gln Gly Ser Lys Gln Asp Val Asp Ala Leu Val 740 745 750 Gln Tyr Ile Tyr Asp Glu Pro Lys Lys Gly Gly Leu Gly Trp Asp Leu 755 760 765 Asp Ala Ile Ile Pro Phe Ala Ala Ile Pro Glu Asn Gly Asn Gly Leu 770 775 780 Asp Asn Ile Asp Ser Lys Ser Glu Phe Ala His Arg Ile Met Leu Thr 785 790 795 800 Asn Leu Leu Arg Leu Leu Gly Ala Val Lys Ser Lys Lys Pro Thr Asp 805 810 815 Thr Arg Pro Ala Gln Cys Ile Leu Pro Leu Ser Pro Asn His Gly Thr 820 825 830 Phe Gly Phe Asp Gly Leu Tyr Ser Glu Ser Lys Ile Ser Leu Glu Thr 835 840 845 Leu Phe Asn Arg Trp Tyr Ser Glu Asp Trp Gly Ser Lys Leu Thr Val 850 855 860 Cys Gly Ala Val Ile Gly Trp Thr Arg Gly Thr Gly Leu Met Ser Ala 865 870 875 880 Asn Asn Ile Ile Ala Glu Gly Ile Glu Lys Leu Gly Val Arg Thr Phe 885 890 895 Ser Gln Lys Glu Met Ala Phe Asn Ile Leu Gly Leu Leu Thr Pro Glu 900 905 910 Ile Val Gln Leu Cys Gln Glu Glu Pro Val Met Ala Asp Leu Asn Gly 915 920 925 Gly Leu Gln Phe Ile Asp Asn Leu Lys Asp Phe Thr Ser Lys Leu Arg 930 935 940 Thr Asp Leu Leu Glu Thr Ala Asp Ile Arg Arg Ala Val Ser Ile Glu 945 950 955 960 Ser Ala Ile Glu Gln Lys Val Val Asn Gly Asp Asn Val Asp Ala Asn 965 970 975 Tyr Ser Lys Val Met Val Glu Pro Arg Ala Asn Met Lys Phe Asp Phe 980 985 990 Pro Thr Leu Lys Ser Tyr Asp Glu Ile Lys Gln Ile Ala Pro Glu Leu 995 1000 1005 Glu Gly Met Leu Asp Leu Glu Asn Val Val Val Val Thr Gly Phe 1010 1015 1020 Ala Glu Val Gly Pro Trp Gly Asn Ser Arg Thr Arg Trp Glu Met 1025 1030 1035 Glu Ala Tyr Gly Glu Phe Ser Leu Glu Gly Ala Ile Glu Met Ala 1040 1045 1050 Trp Ile Met Gly Phe Ile Lys Tyr His Asn Gly Asn Leu Gln Gly 1055 1060 1065 Lys Pro Tyr Ser Gly Trp Val Asp Ala Lys Thr Gln Thr Pro Ile 1070 1075 1080 Asp Glu Lys Asp Ile Lys Ser Lys Tyr Glu Glu Glu Ile Leu Glu 1085 1090 1095 His Ser Gly Ile Arg Leu Ile Glu Pro Glu Leu Phe Asn Gly Tyr 1100 1105 1110 Asp Pro Lys Lys Lys Gln Met Ile Gln Glu Ile Val Val Gln His 1115 1120 1125 Asp Leu Glu Pro Phe Glu Cys Ser Lys Glu Thr Ala Glu Gln Tyr 1130 1135 1140 Lys His Glu His Gly Glu Lys Cys Glu Ile Phe Glu Ile Glu Glu 1145 1150 1155 Ser Gly Glu Tyr Thr Val Arg Ile Leu Lys Gly Ala Thr Leu Tyr 1160 1165 1170 Val Pro Lys Ala Leu Arg Phe Asp Arg Leu Val Ala Gly Gln Ile 1175 1180 1185 Pro Thr Gly Trp Asp Ala Arg Thr Tyr Gly Ile Pro Glu Asp Thr 1190 1195 1200 Ile Ser Gln Val Asp Pro Ile Thr Leu Tyr Val Leu Val Ala Thr 1205 1210 1215 Val Glu Ala Leu Leu Ser Ala Gly Ile Thr Asp Pro Tyr Glu Phe 1220 1225 1230 Tyr Lys Tyr Val His Val Ser Glu Val Gly Asn Cys Ser Gly Ser 1235 1240 1245 Gly Met Gly Gly Val Ser Ala Leu Arg Gly Met Phe Lys Asp Arg 1250 1255 1260 Tyr Ala Asp Lys Pro Val Gln Asn Asp Ile Leu Gln Glu Ser Phe 1265 1270 1275 Ile Asn Thr Met Ser Ala Trp Val Asn Met Leu Leu Leu Ser Ser 1280 1285 1290 Ser Gly Pro Ile Lys Thr Pro Val Gly Ala Cys Ala Thr Ala Val 1295 1300 1305 Glu Ser Val Asp Ile Gly Ile Glu Thr Ile Leu Ser Gly Lys Ala 1310 1315 1320 Lys Val Val Leu Val Gly Gly Tyr Asp Asp Phe Gln Glu Glu Gly 1325 1330 1335 Ser Tyr Glu Phe Ala Asn Met Asn Ala Thr Ser Asn Ser Ile Glu 1340 1345 1350 Glu Phe Lys His Gly Arg Thr Pro Lys Glu Met Ser Arg Pro Thr 1355 1360 1365 Thr Thr Thr Arg Asn Gly Phe Met Glu Ala Gln Gly Ser Gly Ile 1370 1375 1380 Gln Val Ile Met Thr Ala Asp Leu Ala Leu Lys Met Gly Val Pro 1385 1390 1395 Ile His Ala Val Leu Ala Met Thr Ala Thr Ala Thr Asp Lys Ile 1400 1405 1410 Gly Arg Ser Val Pro Ala Pro Gly Lys Gly Ile Leu Thr Thr Ala 1415 1420 1425 Arg Glu His His Gly Asn Leu Lys Tyr Pro Ser Pro Leu Leu Asn 1430 1435 1440 Ile Lys Tyr Arg Lys Arg Gln Leu Asn Lys Arg Leu Glu Gln Ile 1445 1450 1455 Lys Ser Trp Glu Glu Thr Glu Leu Ser Tyr Leu Gln Glu Glu Ala 1460 1465 1470 Glu Leu Ala Lys Glu Glu Phe Gly Asp Glu Phe Ser Met His Glu 1475 1480 1485 Phe Leu Lys Glu Arg Thr Glu Glu Val Tyr Arg Glu Ser Lys Arg 1490 1495 1500 Gln Val Ser Asp Ala Lys Lys Gln Trp Gly Asn Ser Phe Tyr Lys 1505 1510 1515 Ser Asp Pro Arg Ile Ala Pro Leu Arg Gly Ala Leu Ala Ala Phe 1520 1525 1530 Asn Leu Thr Ile Asp Asp Ile Gly Val Ala Ser Phe His Gly Thr 1535 1540 1545 Ser Thr Val Ala Asn Asp Lys Asn Glu Ser Ala Thr Ile Asn Asn 1550 1555 1560 Met Met Lys His Leu Gly Arg Ser Glu Gly Asn Pro Val Phe Gly 1565 1570 1575 Val Phe Gln Lys Tyr Leu Thr Gly His Pro Lys Gly Ala Ala Gly 1580 1585 1590 Ala Trp Met Leu Asn Gly Ala Ile Gln Ile Leu Glu Ser Gly Leu 1595 1600 1605 Val Pro Gly Asn Arg Asn Ala Asp Asn Val Asp Lys Leu Leu Glu 1610 1615 1620 Gln Tyr Glu Tyr Val Leu Tyr Pro Ser Arg Ser Ile Gln Thr Asp 1625 1630 1635 Gly Ile Lys Ala Val Ser Val Thr Ser Phe Gly Phe Gly Gln Lys 1640 1645 1650 Gly Ala Gln Ala Val Val Val His Pro Asp Tyr Leu Phe Ala Val 1655 1660 1665 Leu Asp Arg Ser Thr Tyr Glu Glu Tyr Ala Thr Lys Val Ser Ala 1670 1675 1680 Arg Asn Lys Lys Thr Tyr Arg Tyr Met His Asn Ala Ile Thr Arg 1685 1690 1695 Asn Thr Met Phe Val Ala Lys Asp Lys Ala Pro Tyr Ser Asp Glu 1700 1705 1710 Leu Glu Gln Pro Val Tyr Leu Asp Pro Leu Ala Arg Val Glu Glu 1715 1720 1725 Asn Lys Lys Lys Leu Val Phe Ser Asp Lys Thr Ile Gln Ser Asn 1730 1735 1740 Gln Ser Tyr Val Gly Glu Val Ala Gln Lys Thr Ala Lys Ala Leu 1745 1750 1755 Ser Thr Leu Asn Lys Ser Ser Lys Gly Val Gly Val Asp Val Glu 1760 1765 1770 Leu Leu Ser Ala Ile Asn Ile Asp Asn Glu Thr Phe Ile Glu Arg 1775 1780 1785 Asn Phe Thr Gly Asn Glu Val Glu Tyr Cys Leu Asn Thr Ala His 1790 1795 1800 Pro Gln Ala Ser Phe Thr Gly Thr Trp Ser Ala Lys Glu Ala Val 1805 1810 1815 Phe Lys Ala Leu Gly Val Glu Ser Lys Gly Ala Gly Ala Ser Leu 1820 1825 1830 Ile Asp Ile Glu Ile Thr Arg Asp Val Asn Gly Ala Pro Lys Val 1835 1840 1845 Ile Leu His Gly Glu Ala Lys Lys Ala Ala Ala Lys Ala Gly Val 1850 1855 1860 Lys Asn Val Asn Ile Ser Ile Ser His Asp Asp Phe Gln Ala Thr 1865 1870 1875 Ala Val Ala Leu Ser Glu Phe 1880 1885 23 3069 PRT Mycobacterium tuberculosis 23 Met Thr Ile His Glu His Asp Arg Val Ser Ala Asp Arg Gly Gly Asp 1 5 10 15 Ser Pro His Thr Thr His Ala Leu Val Asp Arg Leu Met Ala Gly Glu 20 25 30 Pro Tyr Ala Val Ala Phe Gly Gly Gln Gly Ser Ala Trp Leu Glu Thr 35 40 45 Leu Glu Glu Leu Val Ser Ala Thr Gly Ile Glu Thr Glu Leu Ala Thr 50 55 60 Leu Val Gly Glu Ala Glu Leu Leu Leu Asp Pro Val Thr Asp Glu Leu 65 70 75 80 Ile Val Val Arg Pro Ile Gly Phe Glu Pro Leu Gln Trp Val Arg Ala 85 90 95 Leu Ala Ala Glu Asp Pro Val Pro Ser Asp Lys His Leu Thr Ser Ala 100 105 110 Ala Val Ser Val Pro Gly Val Leu Leu Thr Gln Ile Ala Ala Thr Arg 115 120 125 Ala Leu Ala Arg Gln Gly Met Asp Leu Val Ala Thr Pro Pro Val Ala 130 135 140 Met Ala Gly His Ser Gln Gly Val Leu Ala Val Glu Ala Leu Lys Ala 145 150 155 160 Gly Gly Ala Arg Asp Val Glu Leu Phe Ala Leu Ala Gln Leu Ile Gly 165 170 175 Ala Ala Gly Thr Leu Val Ala Arg Arg Arg Gly Ile Ser Val Leu Gly 180 185 190 Asp Arg Pro Pro Met Val Ser Val Thr Asn Ala Asp Pro Glu Arg Ile 195 200 205 Gly Arg Leu Leu Asp Glu Phe Ala Gln Asp Val Arg Thr Val Leu Pro 210 215 220 Pro Val Leu Ser Ile Arg Asn Gly Arg Arg Ala Val Val Ile Thr Gly 225 230 235 240 Thr Pro Glu Gln Leu Ser Arg Phe Glu Leu Tyr Cys Arg Gln Ile Ser 245 250 255 Glu Lys Glu Glu Ala Asp Arg Lys Asn Lys Val Arg Gly Gly Asp Val 260 265 270 Phe Ser Pro Val Phe Glu Pro Val Gln Val Glu Val Gly Phe His Thr 275 280 285 Pro Arg Leu Ser Asp Gly Ile Asp Ile Val Ala Gly Trp Ala Glu Lys 290 295 300 Ala Gly Leu Asp Val Ala Leu Ala Arg Glu Leu Ala Asp Ala Ile Leu 305 310 315 320 Ile Arg Lys Val Asp Trp Val Asp Glu Ile Thr Arg Val His Ala Ala 325 330 335 Gly Ala Arg Trp Ile Leu Asp Leu Gly Pro Gly Asp Ile Leu Thr Arg 340 345 350 Leu Thr Ala Pro Val Ile Arg Gly Leu Gly Ile Gly Ile Val Pro Ala 355 360 365 Ala Thr Arg Gly Gly Gln Arg Asn Leu Phe Thr Val Gly Ala Thr Pro 370 375 380 Glu Val Ala Arg Ala Trp Ser Ser Tyr Ala Pro Thr Val Val Arg Leu 385 390 395 400 Pro Asp Gly Arg Val Lys Leu Ser Thr Lys Phe Thr Arg Leu Thr Gly 405 410 415 Arg Ser Pro Ile Leu Leu Ala Gly Met Thr Pro Thr Thr Val Asp Ala 420 425 430 Lys Ile Val Ala Ala Ala Ala Asn Ala Gly His Trp Ala Glu Leu Ala 435 440 445 Gly Gly Gly Gln Val Thr Glu Glu Ile Phe Gly Asn Arg Ile Glu Gln 450 455 460 Met Ala Gly Leu Leu Glu Pro Gly Arg Thr Tyr Gln Phe Asn Ala Leu 465 470 475 480 Phe Leu Asp Pro Tyr Leu Trp Lys Leu Gln Val Gly Gly Lys Arg Leu 485 490 495 Val Gln Lys Ala Arg Gln Ser Gly Ala Ala Ile Asp Gly Val Val Ile 500 505 510 Ser Ala Gly Ile Pro Asp Leu Asp Glu Ala Val Glu Leu Ile Asp Glu 515 520 525 Leu Gly Asp Ile Gly Ile Ser His Val Val Phe Lys Pro Gly Thr Ile 530 535 540 Glu Gln Ile Arg Ser Val Ile Arg Ile Ala Thr Glu Val Pro Thr Lys 545 550 555 560 Pro Val Ile Met His Val Glu Gly Gly Arg Ala Gly Gly His His Ser 565 570 575 Trp Glu Asp Leu Asp Asp Leu Leu Leu Ala Thr Tyr Ser Glu Leu Arg 580 585 590 Ser Arg Ala Asn Ile Thr Val Cys Val Gly Gly Gly Ile Gly Thr Pro 595 600 605 Arg Arg Ala Ala Glu Tyr Leu Ser Gly Arg Trp Ala Gln Ala Tyr Gly 610 615 620 Phe Pro Leu Met Pro Ile Asp Gly Ile Leu Val Gly Thr Ala Ala Met 625 630 635 640 Ala Thr Lys Glu Ser Thr Thr Ser Pro Ser Val Lys Arg Met Leu Val 645 650 655 Asp Thr Gln Gly Thr Asp Gln Trp Ile Ser Ala Gly Lys Ala Gln Gly 660 665 670 Gly Met Ala Ser Ser Arg Ser Gln Leu Gly Ala Asp Ile His Glu Ile 675 680 685 Asp Asn Ser Ala Ser Arg Cys Gly Arg Leu Leu Asp Glu Val Ala Gly 690 695 700 Asp Ala Glu Ala Val Ala Glu Arg Arg Asp Glu Ile Ile Ala Ala Met 705 710 715 720 Ala Lys Thr Ala Lys Pro Tyr Phe Gly Asp Val Ala Asp Met Thr Tyr 725 730 735 Leu Gln Trp Leu Arg Arg Tyr Val Glu Leu Ala Ile Gly Glu Gly Asn 740 745 750 Ser Thr Ala Asp Thr Ala Ser Val Gly Ser Pro Trp Leu Ala Asp Thr 755 760 765 Trp Arg Asp Arg Phe Glu Gln Met Leu Gln Arg Ala Glu Ala Arg Leu 770 775 780 His Pro Gln Asp Phe Gly Pro Ile Gln Thr Leu Phe Thr Asp Ala Gly 785 790 795 800 Leu Leu Asp Asn Pro Gln Gln Ala Ile Ala Ala Leu Leu Ala Arg Tyr 805 810 815 Pro Asp Ala Glu Thr Val Gln Leu His Pro Ala Asp Val Pro Phe Phe 820 825 830 Val Thr Leu Cys Lys Thr Leu Gly Lys Pro Val Asn Phe Val Pro Val 835 840 845 Ile Asp Gln Asp Val Arg Arg Trp Trp Arg Ser Asp Ser Leu Trp Gln 850 855 860 Ala His Asp Ala Arg Tyr Asp Ala Asp Ala Val Cys Ile Ile Pro Gly 865 870 875 880 Thr Ala Ser Val Ala Gly Ile Thr Arg Met Asp Glu Pro Val Gly Glu 885 890 895 Leu Leu Asp Arg Phe Glu Gln Ala Ala Ile Asp Glu Val Leu Gly Ala 900 905 910 Gly Val Glu Pro Lys Asp Val Ala Ser Arg Arg Leu Gly Arg Ala Asp 915 920 925 Val Ala Gly Pro Leu Ala Val Val Leu Asp Ala Pro Asp Val Arg Trp 930 935 940 Ala Gly Arg Thr Val Thr Asn Pro Val His Arg Ile Ala Asp Pro Ala 945 950 955 960 Glu Trp Gln Val His Asp Gly Pro Glu Asn Pro Arg Ala Thr His Ser 965 970 975 Ser Thr Gly Ala Arg Leu Gln Thr His Gly Asp Asp Val Ala Leu Ser 980 985 990 Val Pro Val Ser Gly Thr Trp Val Asp Ile Arg Phe Thr Leu Pro Ala 995 1000 1005 Asn Thr Val Asp Gly Gly Thr Pro Val Ile Ala Thr Glu Asp Ala 1010 1015 1020 Thr Ser Ala Met Arg Thr Val Leu Ala Ile Ala Ala Gly Val Asp 1025 1030 1035 Ser Pro Glu Phe Leu Pro Ala Val Ala Asn Gly Thr Ala Thr Leu 1040 1045 1050 Thr Val Asp Trp His Pro Glu Arg Val Ala Asp His Thr Gly Val 1055 1060 1065 Thr Ala Thr Phe Gly Glu Pro Leu Ala Pro Ser Leu Thr Asn Val 1070 1075 1080 Pro Asp Ala Leu Val Gly Pro Cys Trp Pro Ala Val Phe Ala Ala 1085 1090 1095 Ile Gly Ser Ala Val Thr Asp Thr Gly Glu Pro Val Val Glu Gly 1100 1105 1110 Leu Leu Ser Leu Val His Leu Asp His Ala Ala Arg Val Val Gly 1115 1120 1125 Gln Leu Pro Thr Val Pro Ala Gln Leu Thr Val Thr Ala Thr Ala 1130 1135 1140 Ala Asn Ala Thr Asp Thr Asp Met Gly Arg Val Val Pro Val Ser 1145 1150 1155 Val Val Val Thr Gly Ala Asp Gly Ala Val Ile Ala Thr Leu Glu 1160 1165 1170 Glu Arg Phe Ala Ile Leu Gly Arg Thr Gly Ser Ala Glu Leu Ala 1175 1180 1185 Asp Pro Ala Arg Ala Gly Gly Ala Val Ser Ala Asn Ala Thr Asp 1190 1195 1200 Thr Pro Arg Arg Arg Arg Arg Asp Val Thr Ile Thr Ala Pro Val 1205 1210 1215 Asp Met Arg Pro Phe Ala Val Val Ser Gly Asp His Asn Pro Ile 1220 1225 1230 His Thr Asp Arg Ala Ala Ala Leu Leu Ala Gly Leu Glu Ser Pro 1235 1240 1245 Ile Val His Gly Met Trp Leu Ser Ala Ala Ala Gln His Ala Val 1250 1255 1260 Thr Ala Thr Asp Gly Gln Ala Arg Pro Pro Ala Arg Leu Val Gly 1265 1270 1275 Trp Thr Ala Arg Phe Leu Gly Met Val Arg Pro Gly Asp Glu Val 1280 1285 1290 Asp Phe Arg Val Glu Arg Val Gly Ile Asp Gln Gly Ala Glu Ile 1295 1300 1305 Val Asp Val Ala Ala Arg Val Gly Ser Asp Leu Val Met Ser Ala 1310 1315 1320 Ser Ala Arg Leu Ala Ala Pro Lys Thr Val Tyr Ala Phe Pro Gly 1325 1330 1335 Gln Gly Ile Gln His Lys Gly Met Gly Met Glu Val Arg Ala Arg 1340 1345 1350 Ser Lys Ala Ala Arg Lys Val Trp Asp Thr Ala Asp Lys Phe Thr 1355 1360 1365 Arg Asp Thr Leu Gly Phe Ser Val Leu His Val Val Arg Asp Asn 1370 1375 1380 Pro Thr Ser Ile Ile Ala Ser Gly Val His Tyr His His Pro Asp 1385 1390 1395 Gly Val Leu Tyr Leu Thr Gln Phe Thr Gln Val Ala Met Ala Thr 1400 1405 1410 Val Ala Ala Ala Gln Val Ala Glu Met Arg Glu Gln Gly Ala Phe 1415 1420 1425 Val Glu Gly Ala Ile Ala Cys Gly His Ser Val Gly Glu Tyr Thr 1430 1435 1440 Ala Leu Ala Cys Val Thr Gly Ile Tyr Gln Leu Glu Ala Leu Leu 1445 1450 1455 Glu Met Val Phe His Arg Gly Ser Lys Met His Asp Ile Val Pro 1460 1465 1470 Arg Asp Glu Leu Gly Arg Ser Asn Tyr Arg Leu Ala Ala Ile Arg 1475 1480 1485 Pro Ser Gln Ile Asp Leu Asp Asp Ala Asp Val Pro Ala Phe Val 1490 1495 1500 Ala Gly Ile Ala Glu Ser Thr Gly Glu Phe Leu Glu Ile Val Asn 1505 1510 1515 Phe Asn Leu Arg Gly Ser Gln Tyr Ala Ile Ala Gly Thr Val Arg 1520 1525 1530 Gly Leu Glu Ala Leu Glu Ala Glu Val Glu Arg Arg Arg Glu Leu 1535 1540 1545 Thr Gly Gly Arg Arg Ser Phe Ile Leu Val Pro Gly Ile Asp Val 1550 1555 1560 Pro Phe His Ser Arg Val Leu Arg Val Gly Val Ala Glu Phe Arg 1565 1570 1575 Arg Ser Leu Asp Arg Val Met Pro Arg Asp Ala Asp Pro Asp Leu 1580 1585 1590 Ile Ile Gly Arg Tyr Ile Pro Asn Leu Val Pro Arg Leu Phe Thr 1595 1600 1605 Leu Asp Arg Asp Phe Ile Gln Glu Ile Arg Asp Leu Val Pro Ala 1610 1615 1620 Glu Pro Leu Asp Glu Ile Leu Ala Asp Tyr Asp Thr Trp Leu Arg 1625 1630 1635 Glu Arg Pro Arg Glu Met Ala Arg Thr Val Phe Ile Glu Leu Leu 1640 1645 1650 Ala Trp Gln Phe Ala Ser Pro Val Arg Trp Ile Glu Thr Gln Asp 1655 1660 1665 Leu Leu Phe Ile Glu Glu Ala Ala Gly Gly Leu Gly Val Glu Arg 1670 1675 1680 Phe Val Glu Ile Gly Val Lys Ser Ser Pro Thr Val Ala Gly Leu 1685 1690 1695 Ala Thr Asn Thr Leu Lys Leu Pro Glu Tyr Ala His Ser Thr Val 1700 1705 1710 Glu Val Leu Asn Ala Glu Arg Asp Ala Ala Val Leu Phe Ala Thr 1715 1720 1725 Asp Thr Asp Pro Glu Pro Glu Pro Glu Glu Asp Glu Pro Val Ala 1730 1735 1740 Glu Ser Pro Ala Pro Asp Val Val Ser Glu Ala Ala Pro Val Ala 1745 1750 1755 Pro Ala Ala Ser Ser Ala Gly Pro Arg Pro Asp Asp Leu Val Phe 1760 1765 1770 Asp Ala Ala Asp Ala Thr Leu Ala Leu Ile Ala Leu Ser Ala Lys 1775 1780 1785 Met Arg Ile Asp Gln Ile Glu Glu Leu Asp Ser Ile Glu Ser Ile 1790 1795 1800 Thr Asp Gly Ala Ser Ser Arg Arg Asn Gln Leu Leu Val Asp Leu 1805 1810 1815 Gly Ser Glu Leu Asn Leu Gly Ala Ile Asp Gly Ala Ala Glu Ser 1820 1825 1830 Asp Leu Ala Gly Leu Arg Ser Gln Val Thr Lys Leu Ala Arg Thr 1835 1840 1845 Tyr Lys Pro Tyr Gly Pro Val Leu Ser Asp Ala Ile Asn Asp Gln 1850 1855 1860 Leu Arg Thr Val Leu Gly Pro Ser Gly Lys Arg Pro Gly Ala Ile 1865 1870 1875 Ala Glu Arg Val Lys Lys Thr Trp Glu Leu Gly Glu Gly Trp Ala 1880 1885 1890 Lys His Val Thr Val Glu Val Ala Leu Gly Thr Arg Glu Gly Ser 1895 1900 1905 Ser Val Arg Gly Gly Ala Met Gly His Leu His Glu Gly Ala Leu 1910 1915 1920 Ala Asp Ala Ala Ser Val Asp Lys Val Ile Asp Ala Ala Val Ala 1925 1930 1935 Ser Val Ala Ala Arg Gln Gly Val Ser Val Ala Leu Pro Ser Ala 1940 1945 1950 Gly Ser Gly Gly Gly Ala Thr Ile Asp Ala Ala Ala Leu Ser Glu 1955 1960 1965 Phe Thr Asp Gln Ile Thr Gly Arg Glu Gly Val Leu Ala Ser Ala 1970 1975 1980 Ala Arg Leu Val Leu Gly Gln Leu Gly Leu Asp Asp Pro Val Asn 1985 1990 1995 Ala Leu Pro Ala Ala Pro Asp Ser Glu Leu Ile Asp Leu Val Thr 2000 2005 2010 Ala Glu Leu Gly Ala Asp Trp Pro Arg Leu Val Ala Pro Val Phe 2015 2020 2025 Asp Pro Lys Lys Ala Val Val Phe Asp Asp Arg Trp Ala Ser Ala 2030 2035 2040 Arg Glu Asp Leu Val Lys Leu Trp Leu Thr Asp Glu Gly Asp Ile 2045 2050 2055 Asp Ala Asp Trp Pro Arg Leu Ala Glu Arg Phe Glu Gly Ala Gly 2060 2065 2070 His Val Val Ala Thr Gln Ala Thr Trp Trp Gln Gly Lys Ser Leu 2075 2080 2085 Ala Ala Gly Arg Gln Ile His Ala Ser Leu Tyr Gly Arg Ile Ala 2090 2095 2100 Ala Gly Ala Glu Asn Pro Glu Pro Gly Arg Tyr Gly Gly Glu Val 2105 2110 2115 Ala Val Val Thr Gly Ala Ser Lys Gly Ser Ile Ala Ala Ser Val 2120 2125 2130 Val Ala Arg Leu Leu Asp Gly Gly Ala Thr Val Ile Ala Thr Thr 2135 2140 2145 Ser Lys Leu Asp Glu Glu Arg Leu Ala Phe Tyr Arg Thr Leu Tyr 2150 2155 2160 Arg Asp His Ala Arg Tyr Gly Ala Ala Leu Trp Leu Val Ala Ala 2165 2170 2175 Asn Met Ala Ser Tyr Ser Asp Val Asp Ala Leu Val Glu Trp Ile 2180 2185 2190 Gly Thr Glu Gln Thr Glu Ser Leu Gly Pro Gln Ser Ile His Ile 2195 2200 2205 Lys Asp Ala Gln Thr Pro Thr Leu Leu Phe Pro Phe Ala Ala Pro 2210 2215 2220 Arg Val Val Gly Asp Leu Ser Glu Ala Gly Ser Arg Ala Glu Met 2225 2230 2235 Glu Met Lys Val Leu Leu Trp Ala Val Gln Arg Leu Ile Gly Gly 2240 2245 2250 Leu Ser Thr Ile Gly Ala Glu Arg Asp Ile Ala Ser Arg Leu His 2255 2260 2265 Val Val Leu Pro Gly Ser Pro Asn Arg Gly Met Phe Gly Gly Asp 2270 2275 2280 Gly Ala Tyr Gly Glu Ala Lys Ser Ala Leu Asp Ala Val Val Ser 2285 2290 2295 Arg Trp His Ala Glu Ser Ser Trp Ala Ala Arg Val Ser Leu Ala 2300 2305 2310 His Ala Leu Ile Gly Trp Thr Arg Gly Thr Gly Leu Met Gly His 2315 2320 2325 Asn Asp Ala Ile Val Ala Ala Val Glu Glu Ala Gly Val Thr Thr 2330 2335 2340 Tyr Ser Thr Asp Glu Met Ala Ala Leu Leu Leu Asp Leu Cys Asp 2345 2350 2355 Ala Glu Ser Lys Val Ala Ala Ala Arg Ser Pro Ile Lys Ala Asp 2360 2365 2370 Leu Thr Gly Gly Leu Ala Glu Ala Asn Leu Asp Met Ala Glu Leu 2375 2380 2385 Ala Ala Lys Ala Arg Glu Gln Met Ser Ala Ala Ala Ala Val Asp 2390 2395 2400 Glu Asp Ala Glu Ala Pro Gly Ala Ile Ala Ala Leu Pro Ser Pro 2405 2410 2415 Pro Arg Gly Phe Thr Pro Ala Pro Pro Pro Gln Trp Asp Asp Leu 2420 2425 2430 Asp Val Asp Pro Ala Asp Leu Val Val Ile Val Gly Gly Ala Glu 2435 2440 2445 Ile Gly Pro Tyr Gly Ser Ser Arg Thr Arg Phe Glu Met Glu Val 2450 2455 2460 Glu Asn Glu Leu Ser Ala Ala Gly Val Leu Glu Leu Ala Trp Thr 2465 2470 2475 Thr Gly Leu Ile Arg Trp Glu Asp Asp Pro Gln Pro Gly Trp Tyr 2480 2485 2490 Asp Thr Glu Ser Gly Glu Met Val Asp Glu Ser Glu Leu Val Gln 2495 2500 2505 Arg Tyr His Asp Ala Val Val Gln Arg Val Gly Ile Arg Glu Phe 2510 2515 2520 Val Asp Asp Gly Ala Ile Asp Pro Asp His Ala Ser Pro Leu Leu 2525 2530 2535 Val Ser Val Phe Leu Glu Lys Asp Phe Ala Phe Val Val Ser Ser 2540 2545 2550 Glu Ala Asp Ala Arg Ala Phe Val Glu Phe Asp Pro Glu His Thr 2555 2560 2565 Val Ile Arg Pro Val Pro Asp Ser Thr Asp Trp Gln Val Ile Arg 2570 2575 2580 Lys Ala Gly Thr Glu Ile Arg Val Pro Arg Lys Thr Lys Leu Ser 2585 2590 2595 Arg Val Val Gly Gly Gln Ile Pro Thr Gly Phe Asp Pro Thr Val 2600 2605 2610 Trp Gly Ile Ser Ala Asp Met Ala Gly Ser Ile Asp Arg Leu Ala 2615 2620 2625 Val Trp Asn Met Val Ala Thr Val Asp Ala Phe Leu Ser Ser Gly 2630 2635 2640 Phe Ser Pro Ala Glu Val Met Arg Tyr Val His Pro Ser Leu Val 2645 2650 2655 Ala Asn Thr Gln Gly Thr Gly Met Gly Gly Gly Thr Ser Met Gln 2660 2665 2670 Thr Met Tyr His Gly Asn Leu Leu Gly Arg Asn Lys Pro Asn Asp 2675 2680 2685 Ile Phe Gln Glu Val Leu Pro Asn Ile Ile Ala Ala His Val Val 2690 2695 2700 Gln Ser Tyr Val Gly Ser Tyr Gly Ala Met Ile His Pro Val Ala 2705 2710 2715 Ala Cys Ala Thr Ala Ala Val Ser Val Glu Glu Gly Val Asp Lys 2720 2725 2730 Ile Arg Leu Gly Lys Ala Gln Leu Val Val Ala Gly Gly Leu Asp 2735 2740 2745 Asp Leu Thr Leu Glu Gly Ile Ile Gly Phe Gly Asp Met Ala Ala 2750 2755 2760 Thr Ala Asp Thr Ser Met Met Cys Gly Arg Gly Ile His Asp Ser 2765 2770 2775 Lys Phe Ser Arg Pro Asn Asp Arg Arg Arg Leu Gly Phe Val Glu 2780 2785 2790 Ala Gln Gly Gly Gly Thr Ile Leu Leu Ala Arg Gly Asp Leu Ala 2795 2800 2805 Leu Arg Met Gly Leu Pro Val Leu Ala Val Val Ala Phe Ala Gln 2810 2815 2820 Ser Phe Gly Asp Gly Val His Thr Ser Ile Pro Ala Pro Gly Leu 2825 2830 2835 Gly Ala Leu Gly Ala Gly Arg Gly Gly Lys Asp Ser Pro Leu Ala 2840 2845 2850 Arg Ala Leu Ala Lys Leu Gly Val Ala Ala Asp Asp Val Ala Val 2855 2860 2865 Ile Ser Lys His Asp Thr Ser Thr Leu Ala Asn Asp Pro Asn Glu 2870 2875 2880 Thr Glu Leu His Glu Arg Leu Ala Asp Ala Leu Gly Arg Ser Glu 2885 2890 2895 Gly Ala Pro Leu Phe Val Val Ser Gln Lys Ser Leu Thr Gly His 2900 2905 2910 Ala Lys Gly Gly Ala Ala Val Phe Gln Met Met Gly Leu Cys Gln 2915 2920 2925 Ile Leu Arg Asp Gly Val Ile Pro Pro Asn Arg Ser Leu Asp Cys 2930 2935 2940 Val Asp Asp Glu Leu Ala Gly Ser Ala His Phe Val Trp Val Arg 2945 2950 2955 Asp Thr Leu Arg Leu Gly Gly Lys Phe Pro Leu Lys Ala Gly Met 2960 2965 2970 Leu Thr Ser Leu Gly Phe Gly His Val Ser Gly Leu Val Ala Leu 2975 2980 2985 Val His Pro Gln Ala Phe Ile Ala Ser Leu Asp Pro Ala Gln Arg 2990 2995 3000 Ala Asp Tyr Gln Arg Arg Ala Asp Ala Arg Leu Leu Ala Gly Gln 3005 3010 3015 Arg Arg Leu Ala Ser Ala Ile Ala Gly Gly Ala Pro Met Tyr Gln 3020 3025 3030 Arg Pro Gly Asp Arg Arg Phe Asp His His Ala Pro Glu Arg Pro 3035 3040 3045 Gln Glu Ala Ser Met Leu Leu Asn Pro Ala Ala Arg Leu Gly Asp 3050 3055 3060 Gly Glu Ala Tyr Ile Gly 3065 24 3076 PRT Mycobacterium tuberculosis 24 Met Thr Ile His Glu His Asp Gln Val Ser Ala Asp Arg Asn Gly Asn 1 5 10 15 Ser Leu His Gly Ser Arg Ala Leu Ala Asp Arg Leu Lys Ala Gly Glu 20 25 30 Pro Tyr Val Val Ala Phe Gly Gly Gln Gly Ser Ala Trp Leu Glu Thr 35 40 45 Leu Glu Glu Leu Val Ser Ser Ala Gly Leu Glu Ala Asp Leu Ala Thr 50 55 60 Leu Val Cys Glu Val Glu Leu Leu Leu Glu Pro Val Ala Lys Glu Leu 65 70 75 80 Val Val Val Arg Pro Ile Gly Phe Glu Pro Leu Gln Trp Val Arg Ala 85 90 95 Leu Leu Ala Glu Asp Leu Val Pro Ser Asp Lys His Leu Thr Ser Ala 100 105 110 Ala Val Ser Val Pro Gly Val Leu Leu Thr Gln Ile Ala Val Gly Arg 115 120 125 Ala Leu Ala Arg Gln Gly Met Asp Leu Ile Ala Thr Pro Pro Val Gly 130 135 140 Ile Val Gly His Ser Gln Gly Val Leu Ala Val Glu Ala Leu Lys Ala 145 150 155 160 Gly Gly Ala Arg Asp Ala Glu Leu Leu Ala Met Ala Gln Leu Ile Gly 165 170 175 Ala Ala Gly Thr Leu Val Ala Arg Arg Arg Gly Ile Ser Val Leu Gly 180 185 190 Asp Arg Pro Pro Met Val Ser Val Thr Asn Ala Asp Pro Glu Arg Ile 195 200 205 Arg Arg Leu Leu Asp Glu Phe Ala Gln Asp Val Arg Thr Val Leu Pro 210 215 220 Pro Val Leu Ser Ile Arg Asn Gly Trp Arg Ser Val Val Ile Thr Gly 225 230 235 240 Thr Pro Glu Gln Leu Ser Arg Phe Glu Arg Tyr Cys Arg Gln Ile Ser 245 250 255 Asp Lys Glu Glu Glu Asp Arg Arg Lys Lys Ile Arg Gly Gly Asp Ile 260 265 270 Phe Ala Pro Val Phe Asp Pro Val Gln Val Glu Ile Gly Phe His Thr 275 280 285 Pro His Leu Ala Asp Gly Ile Gly Ile Val Gly Gly Trp Ala Glu Lys 290 295 300 Val Gly Leu Asp Val Thr Leu Ala Arg Glu Leu Thr Glu Ala Ile Leu 305 310 315 320 Val Arg Gly Val Asp Trp Val Arg Glu Ile Thr Arg Val His Gly Ala 325 330 335 Gly Val Arg Trp Ile Ile Asp Leu Gly Pro Gly Asp Ile Leu Thr Arg 340 345 350 Leu Thr Ala Pro Val Ile Arg Gly Leu Gly Val Gly Ile Val Pro Val 355 360 365 Ala Asn Arg Gly Gly Gln Arg Thr Leu Phe Thr Val Gly Ala Val Pro 370 375 380 Glu Val Val Arg Ala Trp Leu Ser Tyr Ala Pro Thr Val Val Gln Leu 385 390 395 400 Pro Asp Gly Arg Ile Lys Leu Ser Thr Lys Phe Thr Arg Leu Thr Gly 405 410 415 Arg Ser Pro Ile Leu Leu Ala Gly Met Thr Pro Thr Thr Val Asp Ala 420 425 430 Asn Ile Val Ala Ala Ala Ala Asn Ala Gly His Trp Ala Glu Leu Ala 435 440 445 Gly Gly Gly Gln Val Thr Glu Glu Ile Phe Ala Asn Arg Val Glu Gln 450 455 460 Leu Ser Gly Leu Leu Glu Pro Gly Arg Thr Tyr Gln Phe Asn Ala Leu 465 470 475 480 Phe Leu Asp Pro Tyr Leu Trp Lys Leu Gln Val Gly Gly Lys Arg Leu 485 490 495 Val Gln Lys Ala Arg Gln Ser Gly Ala Ala Ile Asp Gly Val Val Ile 500 505 510 Ser Gly Gly Ile Leu Asp Leu Glu Asp Ala Val Glu Leu Ile Glu Glu 515 520 525 Leu Gly Gly Ile Gly Ile Ser Tyr Val Val Phe Lys Pro Gly Thr Ile 530 535 540 Glu Gln Ile Arg Ser Val Ile Arg Ile Ala Thr Glu Met Ser Thr Lys 545 550 555 560 Pro Val Ile Met His Val Glu Gly Gly Arg Ala Gly Gly His His Ser 565 570 575 Trp Glu Asp Leu Asp Asp Leu Leu Leu Ala Thr Tyr Ser Glu Leu Arg 580 585 590 Ser His Ala Asn Ile Thr Val Cys Val Gly Gly Gly Ile Gly Thr Pro 595 600 605 Glu Lys Ala Ala Glu Tyr Leu Ser Gly Arg Trp Ala Gln Ala Tyr Gly 610 615 620 Phe Pro Leu Met Pro Ile Asp Gly Ile Leu Val Gly Thr Ala Ala Met 625 630 635 640 Ala Thr Lys Glu Ala Thr Thr Ser Pro Ser Val Lys Arg Met Leu Val 645 650 655 Glu Thr Gln Gly Thr Asp Gln Trp Ile Gly Ser Gly Lys Ala Gln Gly 660 665 670 Gly Met Ala Ser Ser Arg Ser Gln Leu Gly Ala Asp Ile His Glu Ile 675 680 685 Asp Asn Ala Ala Ser Arg Cys Gly Arg Leu Leu Asp Glu Val Ala Gly 690 695 700 Asp Ala Glu Ala Val Ala Glu Arg Arg Asp Glu Ile Ile Ala Ala Met 705 710 715 720 Ala Asn Thr Ala Lys Pro Tyr Phe Gly Asp Val Ser Glu Met Thr Tyr 725 730 735 Leu Gln Trp Leu Gln Arg Tyr Val Glu Leu Thr Ile Gly Glu Gly Asn 740 745 750 Ser Thr Ala Asp Thr Ala Ser Pro Gly Ser Pro Trp Leu Ala Asp Thr 755 760 765 Trp Arg Asp Arg Phe Gln Lys Met Leu Gln Arg Ala Glu Ser Arg Leu 770 775 780 His Pro Ser Asp Phe Gly Leu Ile Lys Thr Ile Phe Thr Asp Pro Val 785 790 795 800 Leu Leu Glu Lys Pro Asn Gln Ala Ile Ala Ala Leu Leu Lys Tyr Tyr 805 810 815 Pro Asp Ala Glu Thr Val Gln Leu His Pro Ala Asp Ala Pro Phe Phe 820 825 830 Val Met Leu Cys Gln Met Leu Gly Lys Pro Val Asn Phe Val Pro Val 835 840 845 Ile Asp Lys Asp Val Arg Arg Trp Trp Arg Ser Asp Ser Leu Trp Gln 850 855 860 Ala His Asp Ala Arg Tyr Asp Ala Asp Gln Val Cys Ile Ile Pro Gly 865 870 875 880 Ile Ala Ala Val Ala Gly Ile Thr Gln Met Asp Glu Pro Val Gly Glu 885 890 895 Leu Leu Asp Arg Phe Glu Gln Ala Ala Ile Asp Glu Val Leu Ala Gly 900 905 910 Gly Ala Glu Pro Val Val Val Met Ser Arg Arg Leu Gly Arg Ala Asp 915 920 925 Val Ala Gly Pro Leu Ala Val Val Leu Asp Ala Pro Asp Val Leu Trp 930 935 940 Ala Gly Arg Ile Ala Thr Asn Pro Val His Arg Ile Ala Asp Pro Asn 945 950 955 960 Glu Trp Gln Val Asn Gly Asn Leu Ser Ala Thr His Ser Ser Thr Gly 965 970 975 Ala Gln Leu Gln Val Lys Ser Glu Asp Gln Gln Val Val Leu Ser Val 980 985 990 Pro Val Ser Asn Gly Trp Ile Asp Ile Pro Phe Thr Leu Pro Thr Asn 995 1000 1005 Thr Val Asp Gly Gly Ala Leu Leu Val Ser Thr Glu Asp Ala Thr 1010 1015 1020 Ser Ala Met Arg Ala Val Leu Ala Ile Val Ala Gly Val Asp Gly 1025 1030 1035 Pro Glu Leu Leu Ser Pro Val Lys Asp Gly Thr Ala Ile Val Thr 1040 1045 1050 Val Asp Trp Asn Pro Glu Arg Val Ala Asp His Thr Gly Val Thr 1055 1060 1065 Ala Thr Phe Arg Glu Pro Leu Ala Pro Ser Leu Ala Thr Val Pro 1070 1075 1080 Asp Ala Leu Val Gly Ala Cys Trp Pro Ala Val Phe Ser Ala Ile 1085 1090 1095 Gly Ser Ala Val Thr Glu Ala Gly Val Leu Val Val Glu Gly Leu 1100 1105 1110 Leu Asn Leu Leu His Leu Asp His Ala Val Cys Val Val Gly Lys 1115 1120 1125 Leu Pro Thr Val Pro Ala Gln Leu Thr Val Thr Ala Thr Val Ser 1130 1135 1140 Leu Ala Ile Asp Thr Asp Met Gly Arg Val Val Pro Val Ser Val 1145 1150 1155 Thr Ile Arg Asp Thr Thr Gly Ala Asp Gly Ala Val Leu Ala Thr 1160 1165 1170 Leu Glu Glu Arg Phe Val Ile Leu Gly Arg Thr Gly Thr Ala Glu 1175 1180 1185 Leu Thr Gly Pro Val Arg Ala Gly Gly Ala Ile Ser Glu Asn Ala 1190 1195 1200 Thr Asp Thr Pro Arg Arg Arg Arg Arg Asp Val Thr Leu Thr Ala 1205 1210 1215 Pro Ile Asp Met Arg Pro Phe Ala Val Val Ser Gly Asp His Asn 1220 1225 1230 Pro Ile His Thr Asp Arg Thr Ala Ala Leu Leu Ala Gly Leu Glu 1235 1240 1245 Ser Pro Ile Val His Gly Met Trp Leu Ser Ala Ala Ala Gln His 1250 1255 1260 Val Val Met Ala Thr Asp Gly Gln Ala Arg Pro Ala Ala Arg Leu 1265 1270 1275 Ile Gly Trp Thr Ala Arg Phe Leu Gly Met Ala His Pro Gly Asp 1280 1285 1290 Lys Val Asp Phe Arg Val Asp Arg Ile Gly Ile Asp Gln Gly Ala 1295 1300 1305 Glu Ile Leu Glu Val Ser Ala Arg Ile Ser Ser Gly Leu Val Met 1310 1315 1320 Ser Ala Thr Ala Arg Leu Ala Ala Pro Lys Thr Val Tyr Ala Phe 1325 1330 1335 Pro Gly Gln Gly Ile Gln His Lys Gly Met Gly Met Asp Val Arg 1340 1345 1350 Ala Arg Ser Lys Ala Ala Arg Arg Val Trp Asp Asp Ala Asp Lys 1355 1360 1365 Phe Thr Arg Ser Gly Leu Gly Phe Ser Val Leu His Val Val Arg 1370 1375 1380 Asp Asn Pro Thr Asn Ile Thr Ala Asn Gly Val His Tyr His His 1385 1390 1395 Pro Asp Gly Val Leu Tyr Leu Thr Gln Phe Thr Gln Val Ala Met 1400 1405 1410 Ala Thr Val Ala Val Ala Gln Val Ala Glu Met Arg Glu Gln Gly 1415 1420 1425 Ala Phe Val Glu Gly Ala Ile Ala Cys Gly His Ser Val Gly Glu 1430 1435 1440 Tyr Thr Ala Leu Ala Cys Val Met Gly Val Tyr Glu Leu Glu Ala 1445 1450 1455 Leu Leu Glu Thr Val Phe His Arg Gly Ser Lys Met His Asp Ile 1460 1465 1470 Val Leu Arg Asp Glu Leu Gly Arg Ser Asn Tyr Arg Leu Ala Ala 1475 1480 1485 Ile Arg Pro Ser Gln Ile Gly Leu Pro Asp Asp Glu Val Pro Ala 1490 1495 1500 Phe Val Arg Gly Ile Ala Glu Ser Thr Gly Glu Phe Leu Glu Ile 1505 1510 1515 Val Asn Phe Asn Leu Arg Gly Ser Gln Tyr Ala Ile Ala Gly Thr 1520 1525 1530 Val His Gly Leu Glu Ala Leu Glu Ala Glu Val Glu Arg Arg Arg 1535 1540 1545 Glu Leu Thr Gly Gly Arg Arg Ser Phe Ile Leu Val Pro Gly Ile 1550 1555 1560 Asp Val Pro Phe His Ser Arg Val Leu Arg Val Gly Val Ala Glu 1565 1570 1575 Phe Arg Arg Ser Leu Asp Arg Val Leu Pro Gln Asp Gln Asp Pro 1580 1585 1590 Asp Trp Ile Ile Gly Arg Tyr Ile Pro Asn Leu Val Pro Arg Pro 1595 1600 1605 Phe Thr Leu Ala Arg Asp Phe Ile Gln Glu Ile Arg Asp Leu Val 1610 1615 1620 Pro Ala Glu Pro Leu Asp Asp Ile Leu Ala Asp Tyr Asp Thr Trp 1625 1630 1635 Arg Arg Glu Arg Pro Ser Glu Met Ala Arg Arg Val Leu Ile Glu 1640 1645 1650 Leu Leu Ala Trp Gln Phe Ala Ser Pro Val Arg Trp Ile Glu Thr 1655 1660 1665 Gln Asp Leu Leu Phe Thr Glu Glu Ala Ala Gly Gly Leu Gly Val 1670 1675 1680 Glu Arg Phe Val Glu Ile Gly Val Lys Ser Ala Pro Thr Val Ala 1685 1690 1695 Gly Leu Ala Thr Asp Thr Leu Lys Leu Pro Glu Tyr Ser His Asn 1700 1705 1710 Thr Val Glu Val Leu Asn Val Glu Arg Asp Ala Ala Val Leu Phe 1715 1720 1725 Ala Thr Asp Thr Asp Pro Glu Leu Glu Pro Glu Pro Glu Asn Val 1730 1735 1740 Ser Asp Ala Ser Ala Ala Leu Pro Ala Glu Ser Ala Leu Ala Leu 1745 1750 1755 Gly Thr Val Ala Pro Ala Pro Val Val Pro Ser Gly Pro Arg Pro 1760 1765 1770 Glu Asp Ile Ser Phe Gly Ala Ala Asp Ala Thr Leu Ala Leu Ile 1775 1780 1785 Ala Leu Ser Ala Lys Met Arg Leu Asp Gln Ile Glu Glu Met Asp 1790 1795 1800 Ser Ile Glu Ser Ile Thr Asp Gly Ala Ser Ser Arg Arg Asn Gln 1805 1810 1815 Leu Leu Val Asp Leu Gly Ser Glu Leu Ser Leu Gly Ala Ile Asp 1820 1825 1830 Gly Val Ala Glu Ala Asp Leu Ala Gly Leu Arg Ser Gln Val Thr 1835 1840 1845 Lys Leu Ala Arg Thr Tyr Lys Pro Tyr Gly Pro Val Leu Ser Glu 1850 1855 1860 Leu Ile Asn Asp Gln Leu Arg Ser Ala Leu Gly Pro Ser Gly Lys 1865 1870 1875 Arg Pro Gly Val Ile Ala Glu Arg Val Lys Lys Ile Trp Glu Leu 1880 1885 1890 Gly Asp Gly Trp Val Lys His Val Thr Val Glu Ile Ala Leu Gly 1895 1900 1905 Thr Arg Glu Gly Thr Ser Val Arg Gly Gly Pro Leu Gly Asn Leu 1910 1915 1920 Asn Glu Gly Ala Leu Ala Asp Val Asp Ser Val Asp Lys Ala Val 1925 1930 1935 Asp Ala Ala Val Ala Ser Val Ala Ala Arg His Gly Val Val Val 1940 1945 1950 Ala Leu Pro Ser Ala Gly Ser Gly Gly Ser Ala Thr Val Asp Val 1955 1960 1965 Ala Ala Leu Ser Glu Phe Thr Asp Gln Ile Thr Gly His Asp Gly 1970 1975 1980 Val Leu Ala Ser Ala Ala Arg Leu Val Leu Gly Gln Leu Gly Leu 1985 1990 1995 Asp Gly Pro Val Thr Ala Ala Pro Ala Thr Thr Asp Thr Gly Leu 2000 2005 2010 Ile Asp Leu Val Thr Ala Glu Leu Ser Thr Asp Trp Pro Arg Leu 2015 2020 2025 Val Ala Pro Val Phe Asp Val Lys Lys Ala Val Val Phe Asp Asp 2030 2035 2040 Arg Trp Ala Ser Ala Arg Glu Asp Leu Val Arg Leu Trp Leu Asn 2045 2050 2055 Asp Glu Gly Glu Ile Glu Ala Gln Trp Ser His Leu Ser Glu Arg 2060 2065 2070 Phe Glu Gly Ala Gly His Val Val Ala Thr Gln Ala Thr Trp Trp 2075 2080 2085 Gln Gly Lys Ser Leu Ala Ala Gly Arg Gln Ile His Ala Ser Leu 2090 2095 2100 Tyr Gly Arg Ile Ala Ala Gly Ala Gln Asn Pro Asp Arg Gly Leu 2105 2110 2115 Tyr Ser Ser Glu Ile Ala Val Val Thr Gly Ala Ser Lys Gly Ser 2120 2125 2130 Ile Ala Ala Ser Val Ala Ala Arg Leu Leu Asp Gly Gly Ala Thr 2135 2140 2145 Val Ile Ala Thr Thr Ser Lys Leu Asp Glu Glu Arg Ile Thr Phe 2150 2155 2160 Tyr Arg Ala Leu Tyr Arg Asp His Ala Arg Tyr Gly Ala Ala Leu 2165 2170 2175 Trp Val Val Ala Ala Asn Met Ala Ser Tyr Ser Asp Ile Asp Ala 2180 2185 2190 Leu Val Glu Trp Ile Gly Asn Glu Gln Thr Glu Ser Leu Gly Pro 2195 2200 2205 Gln Ser Ile His Ile Lys Asp Ala Gln Thr Pro Thr Leu Leu Phe 2210 2215 2220 Pro Phe Ala Ala Pro Arg Val Ile Gly Asp Leu Ser Glu Ala Gly 2225 2230 2235 Ala Arg Ser Glu Ile Glu Met Lys Val Leu Leu Trp Ala Val Gln 2240 2245 2250 Arg Leu Ile Val Gly Leu Ser Lys Ile Gly Thr Glu Arg Asp Val 2255 2260 2265 Ala Ser Arg Leu His Val Val Leu Pro Gly Ser Pro Asn Arg Gly 2270 2275 2280 Met Phe Gly Gly Asp Gly Ala Tyr Gly Glu Ala Lys Ser Ala Leu 2285 2290 2295 Asp Ala Val Val Ser Arg Trp His Ala Glu Ser Ser Trp Ala Ala 2300 2305 2310 Arg Val Ser Leu Ala His Ala Leu Ile Gly Trp Thr Arg Gly Thr 2315 2320 2325 Gly Leu Met Gly His Asn Asp Val Ile Val Ser Ala Val Glu Glu 2330 2335 2340 Ala Gly Val Thr Thr Tyr Ser Thr Asp Glu Met Ala Ala Met Leu 2345 2350 2355 Leu Asp Leu Cys Asn Ala Glu Ser Lys Val Ala Ala Ala Gly Thr 2360 2365 2370 Pro Ile Thr Val Asp Leu Thr Gly Gly Leu Gly Glu Val Asp Leu 2375 2380 2385 Asp Met Ala Glu Leu Ala Ala Lys Ala Arg Glu Asp His Ala Ala 2390 2395 2400 Gln Ala Ala Glu Asp Glu Ala Thr Glu Ala Ser Glu Val Ala Gly 2405 2410 2415 Thr Ile Ala Ala Leu Pro Ser Pro Pro Arg Gly Tyr Thr Pro Ala 2420 2425 2430 Ser Pro His Trp Asp Asp Leu Asp Val Asp Pro Ala Asp Leu Val 2435 2440 2445 Val Ile Val Gly Gly Ala Glu Ile Gly Pro Tyr Gly Ser Ser Arg 2450 2455 2460 Thr Arg Phe Glu Met Glu Val Ala Gly Glu Leu Ser Ala Ala Gly 2465 2470 2475 Val Leu Glu Leu Val Trp Thr Thr Gly Leu Ile Arg Trp Glu Asp 2480 2485 2490 Asp Pro Gln Pro Gly Trp Tyr Asp Thr Glu Ser Gly Glu Leu Val 2495 2500 2505 Asp Glu Ser Glu Leu Val Glu Arg Tyr His Asp Thr Val Val Gln 2510 2515 2520 Arg Cys Gly Ile Arg Glu Phe Val Asp Asp Gly Thr Ile Asp Pro 2525 2530 2535 Asp His Ala Tyr Pro Leu Leu Val Ser Val Phe Leu Asp Lys Asp 2540 2545 2550 Phe Ala Phe Val Val Ser Ser Glu Ala Asp Ala Arg Ala Phe Val 2555 2560 2565 Glu Phe Asp Pro Glu His Thr Val Ile Arg Pro Val Pro Asp Ser 2570 2575 2580 Ser Asp Trp Gln Val Ile Arg Lys Ala Gly Thr Glu Ile Arg Val 2585 2590 2595 Pro Arg Lys Met Lys Leu Ser Arg Val Val Gly Gly Gln Ile Pro 2600 2605 2610 Thr Gly Phe Asp Pro Thr Val Trp Gly Ile Ser Pro Asp Met Val 2615 2620 2625 Ser Ser Ile Asp Arg Val Ala Val Trp Ser Ile Val Ala Thr Val 2630 2635 2640 Asp Ala Phe Leu Ser Ala Gly Phe Thr Pro Ala Glu Val Met Arg 2645 2650 2655 Tyr Val His Pro Ser Leu Val Ala Asn Thr Met Gly Thr Gly Met 2660 2665 2670 Gly Gly Gly Thr Ser Ile Gln Arg Leu Tyr His Ser Ser Leu Leu 2675 2680 2685 Gly Arg Asn Lys Pro Asn Asp Ile Phe Gln Glu Ile Leu Pro Asn 2690 2695 2700 Ile Val Ala Ala His Val Val Gln Ser Tyr Ile Gly Ser Tyr Gly 2705 2710 2715 Ser Met Ile His Pro Val Ala Ala Cys Ala Thr Ala Ala Val Ser 2720 2725 2730 Val Glu Glu Gly Val Asp Lys Ile Arg Leu Gly Lys Ala Glu Leu 2735 2740 2745 Val Val Ala Gly Gly Ile Asp Asp Leu Thr Leu Glu Gly Ile Ile 2750 2755 2760 Gly Phe Gly Asp Met Ala Ala Thr Ala Asp Thr Ala Met Met Arg 2765 2770 2775 Gly Arg Gly Ile His Asp Ser Lys Phe Ser Arg Pro Asn Asp Arg 2780 2785 2790 Arg Arg Leu Gly Phe Val Glu Ala Gln Gly Gly Gly Thr Ile Leu 2795 2800 2805 Leu Ala Arg Gly Asp Leu Ala Leu Lys Met Gly Leu Pro Val Phe 2810 2815 2820 Ala Val Val Ala Phe Ala Gln Ser Phe Gly Asp Gly Val His Thr 2825 2830 2835 Ser Ile Pro Ala Pro Gly Leu Gly Ala Leu Gly Ala Gly Arg Gly 2840 2845 2850 Gly Lys Asp Ser Pro Leu Val Gln Ser Leu Ala Lys Leu Gly Val 2855 2860 2865 Ser Ala Asp Asp Ile Ala Val Ile Ser Lys His Asp Thr Ser Thr 2870 2875 2880 Leu Ala Asn Asp Pro Asn Glu Thr Glu Leu His Glu Arg Leu Ala 2885 2890 2895 Asp Ala Met Gly Arg Ser Ala Gly Ala Pro Leu Phe Val Val Ser 2900 2905 2910 Gln Lys Ser Leu Thr Gly His Ala Lys Gly Gly Ala Ala Val Phe 2915 2920 2925 Gln Met Met Gly Leu Cys Gln Met Leu Arg Asp Gly Val Ile Pro 2930 2935 2940 Pro Asn Arg Ser Leu Asp Cys Val Asp Glu Glu Leu Ala Gly Ala 2945 2950 2955 Ala His Phe Val Trp Leu Arg Asp Thr Leu Arg Leu Gly Glu Lys 2960 2965 2970 Phe Pro Leu Lys Ala Gly Met Leu Thr Ser Leu Gly Phe Gly His 2975 2980 2985 Val Ser Gly Leu Val Ala Leu Val His Pro Gln Ala Phe Ile Ala 2990 2995 3000 Ala Leu Asp Pro Gly Gln Arg Asp Asp Tyr Gln Arg Arg Ala Asn 3005 3010 3015 Val Arg Leu Leu Ala Gly Gln Arg Arg Leu Ala Ser Ala Ile Ala 3020 3025 3030 Gly Gly Ala Pro Met Tyr Glu Arg Pro Pro Asp Arg Arg Phe Asp 3035 3040 3045 His His Val Pro Glu Lys Leu Gln Glu Ala Ala Met Leu Leu Asn 3050 3055 3060 Pro Ala Ala Arg Leu Gly Asp Gly Asp Ala Tyr Ile Gly 3065 3070 3075 25 2586 PRT Caenorhabditis elegans 25 Met Asp Pro Thr Gln Trp Trp Gln Lys Gln Asp Asp Ile Val Ile Ser 1 5 10 15 Gly Val Ser Gly Arg Phe Pro Arg Cys Asp Asn Val Lys Met Phe Gly 20 25 30 Asp Met Leu Leu Ala Gly Glu Asp Leu Val Thr Glu Asp Ser Leu Arg 35 40 45 Trp Thr Pro Gly Phe Cys Asp Leu Pro Lys Arg His Gly Lys Leu Lys 50 55 60 Val Leu Asn Lys Phe Asp Ala Gly Phe Phe Gln Val Thr Pro Lys Gln 65 70 75 80 Ala Asn Phe Met Asp Pro Gln Val Arg Leu Leu Leu Glu Ala Ser Trp 85 90 95 Glu Ala Met Val Asp Ala Gly Ile Asn Pro Thr Asp Leu Arg Gly Ser 100 105 110 Lys Thr Gly Val Phe Val Gly Cys Ser Ala Ser Glu Thr Ser Gly Met 115 120 125 Leu Thr Gln Asp Pro Asp Thr Val Thr Gly Tyr Thr Leu Thr Gly Cys 130 135 140 Val Arg Ser Met Phe Ser Asn Arg Ile Ser Tyr Thr Phe Asp Leu Gln 145 150 155 160 Gly Pro Ser Phe Ser Val Asp Thr Ala Cys Ser Ser Ser Leu Leu Ala 165 170 175 Leu Gln Leu Ala Val Asp Ser Ile Arg Gln Gly Gln Cys Asp Ala Ala 180 185 190 Ile Val Ala Gly Ala His Leu Thr Leu Thr Pro Thr Ala Ala Leu Gln 195 200 205 Phe Leu Arg Leu Gly Met Leu Thr Asp Lys Gly Ser Cys Arg Ser Phe 210 215 220 Asp Glu Ser Gly Asp Gly Tyr Cys Arg Thr Glu Gly Val Ala Ala Ile 225 230 235 240 Phe Ile Gln Arg Lys Lys Lys Ala Gln Arg Leu Tyr Ala Thr Val Val 245 250 255 His Ala Lys Ser Asn Thr Asp Gly His Lys Glu His Gly Ile Thr Phe 260 265 270 Pro Ser Gly Glu Arg Gln Ala Gln Leu Leu Gln Glu Val Tyr Ser Glu 275 280 285 Ala Gly Ile Asp Pro Asn Ser Val Tyr Tyr Val Glu Ala His Gly Thr 290 295 300 Gly Thr Lys Val Gly Asp Pro Gln Glu Ala Asn Ala Ile Cys Glu Val 305 310 315 320 Phe Cys Ser Lys Arg Thr Asp Ser Leu Leu Ile Gly Ser Val Lys Ser 325 330 335 Asn Met Gly His Ala Glu Pro Ala Ser Gly Val Cys Ser Leu Thr Lys 340 345 350 Ile Leu Leu Ser Ile Glu Arg Gln Leu Ile Pro Pro Asn Leu His Tyr 355 360 365 Asn Thr Pro Asn Gln Tyr Ile Pro Gly Leu Thr Asp Gly Arg Leu Lys 370 375 380 Val Val Thr Glu Pro Thr Ala Leu Pro Gly Gly Leu Ile Gly Ile Asn 385 390 395 400 Ser Phe Gly Phe Gly Gly Ser Asn Thr His Val Ile Leu Lys Ala Ala 405 410 415 Asp His Ile Ala Pro Pro Ile Thr Pro His Pro Phe Thr Lys Leu Val 420 425 430 Thr Tyr Cys Gly Arg Thr Gln Glu Ala Val Glu Asn Ile Phe Thr Glu 435 440 445 Ile Glu Ser Asn Lys Asp Asp Leu Tyr Leu Gln Ala Leu Leu Ala Asn 450 455 460 Gln Ala Asn Met Pro Ala Asn Leu Leu Pro Phe Arg Gly Tyr Met Leu 465 470 475 480 Leu Asp Arg Glu Asn Asn Val Glu Thr Leu Lys Ser Ile Thr Lys Val 485 490 495 Pro Ile Thr Glu Ala Arg Pro Ile Tyr Phe Ile Tyr Ser Gly Met Gly 500 505 510 Ser Gln Trp Pro Gly Met Ala Ile Lys Leu Met Lys Ile Pro Met Phe 515 520 525 Asp Asp Ser Leu Arg Ala Ser Ser Lys Thr Leu Glu Glu Phe Gly Leu 530 535 540 Asp Val Tyr Gly Met Leu Cys Asn Pro Asp Pro Glu Gln Tyr Ser Asn 545 550 555 560 Asn Thr Met Asn Cys Met Leu Ala Ile Thr Ala Ile Gln Ile Ala Leu 565 570 575 Thr Asp Val Leu Thr Ala Leu Gly Val Ser Pro Asp Gly Ile Ile Gly 580 585 590 His Ser Thr Gly Glu Met Gly Cys Gly Tyr Ala Asp Gly Gly Ile Thr 595 600 605 Arg Glu Gln Thr Met Arg Leu Ala Tyr His Arg Gly Thr Thr Ile Met 610 615 620 Lys His Thr Glu Ile Lys Gly Ala Met Ala Ala Val Gly Leu Thr Trp 625 630 635 640 Glu Gln Val Lys Glu Gln Ala Pro Pro Gly Val Val Ala Ala Cys His 645 650 655 Asn Gly Ala Asp Ser Val Thr Ile Ser Gly Asp Ala Glu Gly Val Ala 660 665 670 Thr Phe Cys Ala Gln Leu Lys Glu Lys Asp Ile Phe Ala Lys Val Val 675 680 685 Asp Thr Ser Gly Ile Pro Phe His Ser Pro Ala Met Leu Ala Val Gln 690 695 700 Asp Glu Met Ile Glu Cys Met Arg Thr Ala Val Pro Glu Pro Lys Pro 705 710 715 720 Arg Ser Ser Lys Trp Ile Ser Thr Ser Ile Pro Glu Asp Asp Trp Glu 725 730 735 Ser Asp Leu Ala Ala Thr Cys Ser Ala Glu Tyr His Val His Asn Ala 740 745 750 Cys Ser Pro Val Leu Phe Tyr Glu Ala Ile Gln Lys Ile Pro Ala Asn 755 760 765 Ala Val Thr Ile Glu Met Ala Pro His Ser Leu Met Gln Ala Ile Leu 770 775 780 Arg Arg Ser Leu Gln Lys Thr Val Thr Asn Val Gly Leu Met Asn Arg 785 790 795 800 Pro Lys Ser Glu Asn Asp Asp Glu Leu Glu Ser Phe Leu Gly Ser Leu 805 810 815 Gly Lys Ile Tyr Gln Ala Gly Val Asn Ile Gln Ile Thr Glu Leu Tyr 820 825 830 Pro Gly Gly Gln Tyr Lys Gly Val Val Pro Lys Gly Thr Pro Met Ile 835 840 845 Gly Pro Met Trp Lys Trp Asp His Thr Gln Asp Trp Leu Thr Ile Asp 850 855 860 Gly Arg Gln Val Leu Ala Gly Gly Ser Gly Ser Val Ala Ser Ser Ala 865 870 875 880 Thr Tyr Asn Ile Asp Pro Phe Ala Thr Asp Ser Lys Glu Thr Tyr Leu 885 890 895 Leu Asp His Val Ile Asp Gly Arg Val Leu Tyr Pro Phe Thr Gly His 900 905 910 Met Val Leu Ala Trp Arg Thr Leu Cys Lys Leu Lys Gly Leu Asp Tyr 915 920 925 Thr Lys Thr Pro Val Val Phe Glu Asn Ile Asn Val Phe Ser Ala Thr 930 935 940 Ile Leu Thr Lys Pro Ile Lys Leu Asp Val Val Leu Ser Pro Gly Asn 945 950 955 960 Gly Tyr Phe Glu Ile Ile Ser Asp Asp Gln Val Ala Ala Ser Gly Arg 965 970 975 Ile Tyr Ile Pro Glu Asp Asn Gln Pro Phe Tyr Tyr Gly Lys Leu Glu 980 985 990 Asp Ile Arg Thr Ser Glu Ile Ala Asp Arg Ile Glu Leu Asp Thr Glu 995 1000 1005 Asp Ala Tyr Lys Glu Phe Leu Leu Arg Gly Tyr Glu Tyr Gly Gln 1010 1015 1020 Ala Phe Arg Gly Ile Tyr Lys Thr Cys Asn Ser Gly Glu Arg Gly 1025 1030 1035 Phe Leu Tyr Trp Thr Gly Asn Trp Val Thr Phe Leu Asp Ser Leu 1040 1045 1050 Leu Gln Thr Ala Leu Leu Ala Glu Arg Ser Asp Thr Leu Arg Leu 1055 1060 1065 Pro Thr Arg Val Arg His Leu Arg Ile Asp Pro Asn Lys His Leu 1070 1075 1080 Glu His Val Val Glu Lys Asp Gly Ile Gln Val Ile Glu Leu Arg 1085 1090 1095 Asn Asp His Ser Thr Asn Gly Cys Ile Ala Gly Gly Val Glu Cys 1100 1105 1110 Cys Asp Leu Asn Ala His Ser Val Ala Arg Arg Ile Gln Val Ser 1115 1120 1125 Gly Gln Leu Tyr His Glu Lys Ile Phe Phe Val Pro His Phe Asp 1130 1135 1140 His Asn Cys Leu Ser Gly His Lys Lys Thr Ser Thr Ile Leu Lys 1145 1150 1155 Asp Tyr Ser Ala Val Ile Lys Gln Gln Leu Tyr Thr Gly Phe Ser 1160 1165 1170 Lys Trp Gln Ser Ala Gly Leu Leu Lys Lys Leu Lys Asn Gly Ala 1175 1180 1185 Gln Ile Val Lys Ala Leu Ala Val Leu Lys Ala Ser Gln Ser Asp 1190 1195 1200 Val Val Leu Asp Asp Thr Val Thr Arg Phe Thr His Asp Gly Lys 1205 1210 1215 Cys Thr Val Leu His His Ile Ala Asp Met Phe Lys Ile Glu Asp 1220 1225 1230 Cys Glu Asp Phe Glu Asp Arg Val Ala Ala Lys Leu Lys Ser Val 1235 1240 1245 Arg Gly Ile Phe Glu Leu Asp Arg Leu Trp Ala Gly Ala Val Leu 1250 1255 1260 Asn Asp Arg Ile Val Lys Ser Leu Gln Asp Ile Cys Ile Glu Asn 1265 1270 1275 Ser Ala Gly His His Ala Thr Met Ala Ala Val Asp Leu Val Ser 1280 1285 1290 Thr Asp Gln Ile Arg His Cys Ile Glu Ala Asn Ser Ser His Pro 1295 1300 1305 Leu Leu Glu Thr Asp Tyr Thr Cys Ile Gly Ala Asn Val Asp His 1310 1315 1320 Leu Asp Glu Ser Thr Leu Glu Ile Ile Gly Gly Lys Lys Gln Lys 1325 1330 1335 Ile Asp Leu Glu Asn Asn Phe Thr Gly His Gly Glu Val Lys Asn 1340 1345 1350 Leu Asp Tyr Val Leu Leu Asp Lys Val Ile Ser Lys Lys Ala Asp 1355 1360 1365 Pro Ile Ala Phe Ile Glu Ala Cys Lys His Leu Ile Arg Glu Thr 1370 1375 1380 Gly Phe Leu Leu Val Val Glu Val Thr Ser Gln Tyr Glu Ile Ala 1385 1390 1395 Leu Ala Ile Glu Gly Leu Leu Gly Asn Glu Met Val Gly Asp Ala 1400 1405 1410 Ser Arg Lys Tyr Asn Gln Phe Phe Thr His Glu Gln Leu Leu Asp 1415 1420 1425 Met Phe Lys Ser Thr Gly Phe Leu Ile Cys Asn Phe Gln Ser Asp 1430 1435 1440 Pro Ala Leu Met Thr Thr Thr Tyr Ala Val Arg Arg Val Ser Pro 1445 1450 1455 Ile Pro Arg Asp Pro Val Phe Ile Asp Val Asp Asp Val Lys Glu 1460 1465 1470 Phe Asn Trp Ile Glu Pro Leu Gln Lys Val Ser Glu Glu Arg Leu 1475 1480 1485 Asn Glu Pro Asp Ser Lys Thr Ile Trp Leu Val Ser Asn Lys Cys 1490 1495 1500 Arg Asn Asn Gly Ile Val Gly Leu Gly Leu Cys Phe Val Glu Glu 1505 1510 1515 Asn Leu Lys Ile Asn Arg Phe Arg Ser Ala Phe Asp Met Ser Ala 1520 1525 1530 Asn Lys Glu Ile Arg Asp Gly Pro Pro Val Trp Asn Ile Gly Asp 1535 1540 1545 Glu Glu Thr Lys Lys Ile Val Glu Leu Asp Leu His Ala Asn Asp 1550 1555 1560 Tyr Met Asp Gly Gln Trp Gly Ser Met Arg His Ile Val Val Lys 1565 1570 1575 Asp Glu Asp Val His Val Tyr Lys Asp Cys Glu His Ala Phe Ile 1580 1585 1590 Asn Thr Leu Thr Arg Gly Asp Val Ser Ser Leu Thr Trp Phe Glu 1595 1600 1605 Ser Pro Asn Gln Tyr Phe Asp Ser Met Val Lys Ser Lys Ala Thr 1610 1615 1620 Gln Glu Leu Cys Ser Val Tyr Tyr Ala Pro Ile Asn Phe Arg Asp 1625 1630 1635 Ile Met Leu Ala Tyr Gly Arg Leu Pro Pro Asp Ala Ile Pro Gly 1640 1645 1650 Asn Phe Ala Asp Arg Glu Cys Leu Leu Gly Met Glu Phe Ser Gly 1655 1660 1665 Arg Leu Lys Asp Gly Thr Arg Leu Met Gly Ile Leu Pro Ala Gln 1670 1675 1680 Ala Leu Ala Thr Thr Val Met Val Asp Arg Asp Tyr Ala Trp Glu 1685 1690 1695 Val Pro Arg Asp Trp Thr Leu Ala Glu Ala Ser Thr Val Pro Val 1700 1705 1710 Val Tyr Thr Thr Ala Tyr Tyr Ala Leu Val Arg Arg Gly Leu Met 1715 1720 1725 Lys Lys Gly Asp Lys Ile Leu Ile His Gly Gly Ala Gly Gly Val 1730 1735 1740 Gly Gln Ala Ala Ile Ala Ile Ala Leu Ala Ala Gly Cys Glu Val 1745 1750 1755 Phe Thr Thr Val Gly Ser Ala Glu Lys Arg Glu Phe Leu Lys Asn 1760 1765 1770 Leu Phe Pro Gln Leu Gln Glu His His Phe Ala Asn Ser Arg Ser 1775 1780 1785 Ala Asp Phe Glu Leu His Ile Arg Gln His Thr Lys Gly Arg Gly 1790 1795 1800 Val Asn Ile Val Leu Asn Ser Leu Ala Asn Glu Met Leu Gln Ala 1805 1810 1815 Ser Leu Arg Cys Leu Ala Arg His Gly Arg Phe Leu Glu Ile Gly 1820 1825 1830 Lys Val Asp Leu Ser Gln Asn Ser Ser Leu Gly Met Ala Lys Leu 1835 1840 1845 Leu Asp Asn Val Ser Val His Gly Ile Leu Leu Asp Ser Ile Met 1850 1855 1860 Asp Pro Thr Val Gly Asp Leu Asp Glu Trp Lys Glu Ile Ala Arg 1865 1870 1875 Leu Leu Glu Gln Gly Ile Lys Ser Gly Val Val Lys Pro Leu His 1880 1885 1890 Ser His Ser Phe Pro Ala Asp Lys Ala Glu Glu Ala Phe Arg Phe 1895 1900 1905 Met Ser Ala Gly Lys His Ile Gly Lys Val Ile Met Glu Ile Arg 1910 1915 1920 Pro Asp Glu Gly Thr Lys Val Cys Pro Pro Ser Lys Ile Ser Val 1925 1930 1935 Arg Ala Ile Cys Arg Thr Leu Cys His Pro Gln His Thr Tyr Leu 1940 1945 1950 Ile Thr Gly Gly Leu Gly Gly Phe Gly Leu Glu Leu Ala Gln Trp 1955 1960 1965 Leu Ile Asn Arg Gly Ala Arg Lys Leu Val Leu Thr Ser Arg Thr 1970 1975 1980 Gly Ile Arg Thr Gly Tyr Gln Ala Arg Cys Val His Phe Trp Arg 1985 1990 1995 Arg Thr Gly Val Ser Val Leu Val Ser Thr Leu Asn Ile Ala Lys 2000 2005 2010 Lys Ser Asp Ala Val Glu Leu Ile Asn Gln Cys Thr Ala Met Gly 2015 2020 2025 Pro Ile Gly Gly Ile Phe His Leu Ala Met Val Leu Arg Asp Cys 2030 2035 2040 Leu Phe Glu Asn Gln Asn Val Gln Asn Phe Lys Asp Ala Ala Glu 2045 2050 2055 Ala Lys Tyr Tyr Gly Thr Ile Asn Leu Asp Tyr Ala Ser Arg Glu 2060 2065 2070 His Cys Asp Lys Asn Ile Leu Lys Trp Phe Val Val Phe Ser Ser 2075 2080 2085 Ile Thr Ser Gly Arg Gly Asn Ala Gly Gln Thr Asn Tyr Gly Trp 2090 2095 2100 Ser Asn Ser Cys Met Glu Arg Met Ile Asp Gln Arg Arg Ala Asp 2105 2110 2115 Gly Phe Pro Gly Ile Ala Ile Gln Trp Gly Ala Ile Gly Asp Val 2120 2125 2130 Gly Val Ile Leu Glu Asn Met Gly Asp Asn Asn Thr Val Val Gly 2135 2140 2145 Gly Thr Leu Pro Gln Arg Met Pro Ser Cys Leu Ser Ser Leu Asp 2150 2155 2160 Asn Phe Leu Ser Trp Asn His Pro Ile Val Ser Ser Phe Ile Lys 2165 2170 2175 Ala Glu Leu Gly Ser Lys Lys Asn Val Gly Gly Gly Asp Leu Met 2180 2185 2190 Ala Thr Ile Ala His Ile Leu Gly Val Asn Asp Ile Ser Gln Leu 2195 2200 2205 Asn Ala Asp Ala Asn Leu Ser Asp Leu Gly Leu Asp Ser Leu Met 2210 2215 2220 Gly Val Glu Ile Lys Gln Ala Leu Glu Arg Asp His Asp Ile Val 2225 2230 2235 Leu Ser Met Lys Glu Ile Arg Thr Leu Thr Leu Asn Lys Leu Gln 2240 2245 2250 Gln Leu Ala Asp Gln Gly Gly Thr Gly Arg Thr Ala Leu Gln Val 2255 2260 2265 Asn Glu Leu Glu Met Lys Lys Asp Gly Glu Arg Asp Ala Glu Leu 2270 2275 2280 Asn Thr Ala Glu Met Leu Glu Gln Gln Met Asn Gln Leu Phe Lys 2285 2290 2295 Met Arg Val Asp Val Asn Asp Leu Asp Pro Gln Asp Ile Ile Val 2300 2305 2310 Lys Ala Asn Lys Val Glu Glu Gly Pro Ile Thr Phe Phe Val His 2315 2320 2325 Ser Ile Glu Gly Ile Ala Thr Pro Leu Lys Lys Val Met Asn Lys 2330 2335 2340 Cys Glu Phe Pro Ala Tyr Cys Phe Gln Ser Thr Lys Asn Val Pro 2345 2350 2355 Gln Thr Ser Ile Glu Asp Val Ala Lys Cys Tyr Ile Arg Glu Met 2360 2365 2370 Lys Lys Ile Gln Pro Ser Gly Pro Tyr Arg Leu Val Gly Tyr Ser 2375 2380 2385 Tyr Gly Ala Cys Ile Gly Phe Glu Met Ala Asn Met Leu Gln Glu 2390 2395 2400 Ser Asp Gly Arg Asp Ala Val Glu Arg Leu Ile Leu Leu Asp Gly 2405 2410 2415 Ser His Leu Tyr Met Gln Thr Tyr Arg Asn Val Tyr Arg Met Ala 2420 2425 2430 Phe Gly Val Thr Gly Asp Ser Leu Val Asn Asn Pro Leu Phe Glu 2435 2440 2445 Ser Glu Ile Met Cys Ala Met Thr Leu Arg Phe Ala Asn Val Asp 2450 2455 2460 Tyr Lys Lys Phe Arg Phe Glu Leu Leu Gln Gln Pro Gly Phe Lys 2465 2470 2475 Ala Arg Val Gln Lys Val Val Asp Gln Val Met Leu Thr Gly Leu 2480 2485 2490 Phe Lys Ser Pro Glu Thr Val Ala Phe Ala Cys Glu Ala Met His 2495 2500 2505 Ser Lys Phe Leu Met Ala Asp Lys Tyr Lys Pro Arg Arg Asn Phe 2510 2515 2520 Gly Gly His Ile Thr Leu Ile Arg Ala Glu Gln Gly Ala Ala Arg 2525 2530 2535 Glu Glu Asp Val Gly Glu Asp Tyr Gly Val Ala Ala Val Ser Glu 2540 2545 2550 Asp Cys Glu Val Leu Lys Val Lys Gly Asp His Asp Thr Phe Val 2555 2560 2565 Gln Gly Lys Ser Ser Ser Val Thr Val Glu His Ile Asn Arg Ile 2570 2575 2580 Ile Leu Gln 2585 26 8936 DNA Rattus norvegicus 26 aggctgggct ctatgggttg cctaagcggt ctggaaagct gaaggatctg tccaagttcg 60 acgcctcctt ttttggggtc caccccaagc aggcacacac aatggacccg cagctccggc 120 tgctgctgga agtcagctat gaagctattg tggacggagg tatcaacccg gcctcactcc 180 gaggaacaaa cactggtgtc tgggtgggtg tgagtggttc cgaggcgtcg gaggccctga 240 gcagagatcc tgagactctt ctgggctaca gcatggtggg ctgccagaga gcaatgatgg 300 ccaaccggct ctctttcttc ttcgacttca aaggacccag cattgccctg gacacagcct 360 gctcctctag cctactggca ctacagaatg cctatcaggc tatccgcagt ggggagtgcc 420 ctgctgccat tgtgggcggg atcaacctgc tgctaaagcc taacacctct gtgcagttca 480 tgaagctagg catgctcagc cccgatggca cctgcagatc ctttgatgat tcagggaacg 540 ggtattgccg tgctgaggct gtcgtggcag ttctgctgac taagaagtcc ttggctcggc 600 gagtctatgc cactattctg aatgccggga cgaacacaga tggctgcaag gagcaaggcg 660 tgacattccc ctctggagaa gcccaggaac aactcatccg ttctctgtat cagccgggcg 720 gtgtggcccc cgagtctctt gaatatattg aagcccatgg cacgggcacc aaggtggggg 780 acccccagga actgaacggc attactcggt ccctgtgtgc tttccgccag agccctttgt 840 taattggctc caccaaatcc aacatgggac accctgagcc tgcctcgggg cttgcagccc 900 tgaccaaggt gctgttatcc ctagaaaatg gggtttgggc ccccaacctg catttccaca 960 accccaaccc tgaaatccca gcacttcttg atgggcggct gcaggtggtc gataggcccc 1020 tgcctgttcg tggtggcatc gtgggcatca actcgtttgg cttcggaggt gccaatgttc 1080 acgtcatcct ccagcccaac acacagcagg ccccagcacc tgccccacat gctgccctac 1140 cgcatttgct gcatgccagt ggacggacca tggaggcagt gcagggcctg ctggaacagg 1200 gccgccagca cagtcaggac ttggcctttg ttagcatgct caatgacatt gcagcaaccc 1260 ctacagcagc catgcccttc agaggttaca ctgtgttagg tgttgagggc catgtccagg 1320 aagtgcagca agtgcctgcc agccagcgcc cactctggtt catctgctca gggatgggca 1380 cacagtggcg tggaatgggg ctgagcctta tgcgcctgga cagtttccgt gagtccatcc 1440 tgcgctctga tgaggctctg aagcccttgg gagtcaaagt gtcagacctg ctgctgagca 1500 ctgatgagca cacctttgat gacatcgtgc attcctttgt gagcctcacc gccatccaga 1560 ttgccctcat cgacctgctg acgtctatgg ggctgaaacc tgatggcatc attgggcact 1620 ccttgggaga ggttgcctgt ggctatgcag atggctgtct ctcccagaga gaggctgtgc 1680 ttgcagccta ctggagaggc cagtgcatta aggatgccaa ccttccggct ggatccatgg 1740 cagctgttgg tttgtcctgg gaagaatgta aacaacgctg ccctcctggt gtggtgcctg 1800 cctgccacaa ctctgaggac actgtgacca tctctggacc tcaggctgca gtgaatgaat 1860 ttgtggagca gctaaagcaa gagggcgtgt ttgccaagga ggtgcgaaca ggtggcctgg 1920 ccttccactc ctacttcatg gaaggaattg cccccacgct gctgcaggct ctcaagaagg 1980 tgatccggga gccacggcca cgctcagcac gctggctcag cacctctatc cctgaggccc 2040 agtggcagag cagcctggcc cgcacatctt ctgctgagta caacgtcaac aacctggtga 2100 gccctgtgct cttccaggaa gcactgtggc acgtccccga gcacgccgtg gtgctggaga 2160 ttgcacccca tgcactgttg caggctgtcc tgaagcgagg cgtgaagcct agctgcacca 2220 tcatcccctt gatgaagagg gaccataaag ataacttgga gttcttcctc accaacctcg 2280 gcaaggtgca cctcacaggc atcgacatca accctaatgc cttgttccca cctgtggaat 2340 tcccggttcc ccgagggact cctctcatct cccctcacat caagtgggac cacagtcaga 2400 cttgggatat cccagttgct gaagacttcc ccaacggttc cagctcctcc tcagctacag 2460 tctacaacat tgacgccagt tccgagtcac ctgaccacta cctggtcgac cactgcattg 2520 acggccgtgt cctcttccct ggcactggct acctgtacct ggtgtggaag acactggctc 2580 gaagcctgag cttgtcccta gaagagaccc ctgtggtgtt tgagaacgtg acatttcatc 2640 aggccaccat cctgcccagg acaggaaccg tgcctctgga ggtgcggctg ctagaggcct 2700 cacatgcatt tgaggtgtct gacagtggca acctgatagt gagcgggaaa gtgtaccagt 2760 gggaagaccc tgactccaag ttattcgacc acccagaagt cccgatcccc gccgagtccg 2820 agtctgtctc ccgcttgacg cagggagaag tatacaagga gctgcggcta cgtggctatg 2880 actatggccc tcatttccag ggcgtctatg aggccaccct cgaaggtgag caaggcaagc 2940 tgctctggaa agacaactgg gtgaccttca tggacacaat gctgcagata tccatcctgg 3000 gcttcagcaa gcagagtctg cagctaccca cccgtgtgac tgccatctat attgaccctg 3060 caacccacct gcagaaggtg tacatgctgg agggagacac tcaagtggct gacgtgacca 3120 cgagccgctg tctgggcgtg accgtctctg gtggtgtcta catttcgaga ctacagacaa 3180 cagcaacctc acggcggcag caggaacagc tggtccccac cctggagaag tttgtcttca 3240 caccccatgt ggagcctgag tgcctgtctg agagtgctat cctgcagaaa gagctgcagc 3300 tgtgcaaggg tctggcaaag gctctgcaga ccaaggccac ccagcaaggg ctgaagatga 3360 cagtgcctgg gctagaggac cttccccagc atggactgcc tcgactcttg gctgctgcct 3420 gccagctgca gctcaacggg aacctgcaac tggagttagg tgaggtactg gctcgagaga 3480 ggctcctgct gccagaagac cctctgatca gtggcctcct taactcccag gccctcaagg 3540 cctgcataga cacagccctg gagaacttgt ctactctcaa gatgaaggtg gtggaggtgc 3600 tggctggaga aggccacttg tattcccaca tctcagcact gctcaacacc cagcctatgc 3660 tgcaactgga gtatacagcc accgaccggc acccccaggc cctgaaggat gttcagacca 3720 agctgcagca gcatgatgta gcacagggcc agtgggaccc ttctggtcct gctcctacca 3780 acctgggtgc tcttgacctt gtggtgtgca actgtgcgtt agccaccctg ggggatccag 3840 ccctggccct ggacaacatg gtagctgccc tcaaggatgg tggtttcctg ctaatgcaca 3900 cagtgctcaa aggacatgcc cttggggaga ccctggcctg cctcccttct gaggtgcagc 3960 ctgggcccag cttcttaagc caggaagagt gggagagcct gttctcaagg aaggcactgc 4020 acctggtggg ccttaaaaag tcattctacg gtactgcgct gttcctgtgc cgccgtctca 4080 gcccacagga caagcccatc ttcctgcctg tggaggatac tagtttccag tgggtggact 4140 ctctgaagag cattctggcc acatcctcct cccagcctgt gtggctaaca gccatgaact 4200 gccccacctc aggtgtggta ggcttggtga actgtctccg aaaagagccg ggtggacacc 4260 ggattcggtg tatcctgctg tccaacctca gcagcacatc tcacgtcccc aagctggacc 4320 ctggctcttc agagctacag aaggtgctag agagtgatct ggtgatgaac gtgtacaggg 4380 acggtgcctg gggtgccttc cgtcacttcc agttagagca ggacaagccc gaggagcaga 4440 cagcacatgc ctttgtaaac gtccttaccc gaggggacct tgcctccatc cgctgggtct 4500 cttctcccct gaaacacatg cagccgccct cgagctcagg agcacagctc tgcactgtct 4560 actatgcctc actgaacttc cgagatatca tgctggccac gggcaagctg tcccctgatg 4620 ccattccagg taaatgggcc agccgggact gcatgcttgg catggagttc tcaggccgtg 4680 ataagtgcgg ccggcgtgtg atggggctgg tacccgcaga aggcctggcc acctcagtcc 4740 tgttatcacc cgacttcctc tgggatgtac cctctagctg gaccctggag gaggcggctt 4800 ctgtgcctgt tgtctacacc accgcctact actccttagt agtgcgtggt cgtattcagc 4860 acggggaaac tgtgctcatt cactcgggct ccggtggtgt gggccaagcg gccatttcca 4920 ttgcccttag cctgggctgc cgagtcttca ccactgtggg ctccgctgag aagcgagctt 4980 acctccaggc cagattccct cagctggatg acaccagctt tgctaactct cgagacacat 5040 cgtttgagca gcatgtgtta ctgcacacag gtggcaaagg ggtggacctg gtcctcaact 5100 ccctggcaga agagaagctg caggccagtg tgcggtgctt ggctcagcat ggccgcttcc 5160 tagagatcgg caaatttgat ctttctaaca accaccctct gggcatggcc atcttcttga 5220 agaacgtcac tttccatggg atcctgctgg atgcactttt tgagggggcc aacgacagct 5280 ggcgggaggt ggcagagctg ctgaaggccg gcatccgtga tggggttgtg aagcctctca 5340 agtgtacagt gtttcccaag gcccaggtgg aggacgcctt ccgatacatg gctcaaggaa 5400 aacatattgg caaagtcctt gtccaggtac gggaggagga gcccgaggct atgctgccag 5460 gggctcagcc caccctgatt tccgccatct ccaagacctt ctgcccagag cataagagtt 5520 acatcatcac tggtggccta ggtggctttg gcctggaact ggcccggtgg cttgtgcttc 5580 gtggggccca aaggcttgta ctaacttccc gatctggaat ccgcacaggc taccaagcca 5640 agcacgttcg ggagtggagg cgccagggca tccatgtgct agtgtcgaca agcaatgtca 5700 gttcactgga gggggcccgt gctctcatcg ctgaagccac aaagcttggg cccgttggag 5760 gtgtcttcaa cctggccatg gttttaaggg atgccatgct ggagaaccag actccagaac 5820 tcttccagga tgtcaacaag cccaagtaca atggcaccct gaaccttgac agggcgaccc 5880 gggaagcctg tcctgagctg gactactttg tggccttctc ctctgtaagc tgcgggcgtg 5940 gtaatgctgg ccaatccaac tatggcttcg ccaactctac catggagcgt atttgcgaac 6000 agcgccggca cgatggcctc ccaggtcttg ccgtgcaatg gggtgccatt ggtgacgtgg 6060 gcattatctt ggaagcgatg ggtaccaatg acacagtcgt tggcggcaca ctgccacagc 6120 gcatctcctc ctgcatggag gtgctggacc tcttcctgaa tcagccccac gcagtcctga 6180 gcagttttgt gctggctgag aagaaagctg tggcccatgg tgatggtgaa gcccagaggg 6240 atctggtgaa agcagtggca cacatcctag gcatccgcga cctcgcaggg attaacctgg 6300 acagctcgct ggcagacctc ggcctggact cgctcatggg tgtggaagtg cgccagatcc 6360 tggaacgtga acatgatctg gtgctaccca ttcgtgaagt acggcaactc acactgcgga 6420 agcttcagga aatgtcctcc aaggctggct cagacactga gttggcagcc cccaagtcca 6480 agaatgatac atccctgaag caggcccagc tgaatctgag tatcctgctg gtgaaccctg 6540 agggccctac cttaacacga ctcaactcag tgcagagctc tgagcggcct ctgttcctgg 6600 tgcaccccat tgaaggttcc atcactgtgt tccacagcct ggctgccaag ctcagtgtgc 6660 ccacctacgg tctgcagtgc acccaagcgg cccccctgga cagcattcca aacctggctg 6720 cctactacat tgattgcatc aagcaggtgc agcctgaggg gccctaccga gtggctgggt 6780 attcttttgg agcttgtgta gccttcgaga tgtgctccca gctgcaggcc cagcagggcc 6840 cagcccccgc ccacaacaac ctcttcttgt ttgatggctc acacacctac gtattggcgt 6900 acacccagag ctaccgggca aagctgaccc caggctgtga ggctgaggct gaagctgaag 6960 ccatatgctt cttcattaag cagtttgttg atgcagagca tagcaaggtg ctagaggccc 7020 tgctaccact gaagagcctg gaggaccggg ttgctgctgc tgtggacctc atcactagaa 7080 gccaccagag cctggaccgc cgtgacctga gctttgctgc cgtgtccttc tactacaagc 7140 ttcgagccgc cgaccagtat aaacccaagg ccaagtacca cggcaatgtg atcctgctgc 7200 gggccaagac aggtggcacc tacggcgagg acttgggtgc cgattacaac ctgtcccagg 7260 tgtgtgatgg gaaggtgtct gtgcacatca ttgagggtga ccaccgtacg ctgctggagg 7320 gcaggggcct ggagtctatc atcaacatca tccacagctc cctggctgag cctcgagtga 7380 gtgtacggga gggctagacc tgcctaccat gaagccacga cccacaccgg ccaccagaga 7440 tgctccgatc cccaccacac cctgagtgca gggactgggg agggtcctgc tggtgggacc 7500 ccctcacccc agtggcccag caccaccccc tcccctggtg gctgctacaa acaggaccat 7560 cacatgtgtc ccagccactt agtggggttc ccagagccac tgacttggag gcaccctggt 7620 ctgtgaagag tcagtggagg ccagcaagag ccaaactgag ccttttctgc caagtgacat 7680 ttgtcacact ggttgtttct ccattaaatt ctcatattta ttgcattgct gggaaagacc 7740 gcccacccca gggttaactc attccagaac ccctaaagtg ggaaaagcca tgtggggaag 7800 gctgctggct ggagcccctt tttgtcttag ccctgtaccc gctcactgca gggcagggta 7860 tggagagggc tggttcgcgg ggaacgagga ccccagcaga cactgtagcc catggccctt 7920 ggtccccagc actcccggct gcacccatga tgcagggcct accagactct gcggaccgca 7980 ccgggcactc actgtatttg ttttccaaga ttcaaattgc tgcttgggtt ttgaatttac 8040 tgcagctgtc agtgtaaaga aacatgtctg aactgtgtcc tttttacacc aacctggtaa 8100 aaatgctctt gatgctgtcc cgttgccaca attaaactgc acgtgagctc tggcttccgt 8160 tcagtctctt tccagtccca gacctgagtc cccagagcct ccacagctct tacagtgaga 8220 atcaaattgg cccactcctt ggaaggcgtg gcattctgtc agagtaaaag gaaagtagag 8280 tgtgctgatt cacgttcagc gtgtggggct ggctagagac cttggcactg tagtgaacag 8340 aatgtgtcca cctttaagtc accctgaagg catcaccata gctacagcct cacccagggg 8400 tagagaatag tactgtctac ttgttgacta cctggcagtt ggtgccagcc cctatagagg 8460 aaaacagcag tgtgtggcca ctgtgagaag catatccctg gaaacaggtg accagagcag 8520 agggctaacg cctacctgag tcacacaaaa ctgaccaggc ttgagtgtcc agaagagtct 8580 atcagaaggc cacagcattc agtcctatcc acagagagca gcagactaag ttgtctcctt 8640 gccagcttag aaaactgcag tgctggggta caggtagggt gttcaggagg tccgggcccc 8700 agtgattagt ctaagactga agcatctggt tggctgtggt cccacctaga aaattcttaa 8760 agctcttgtc atgtacttcc tgggaaggac ctaccctgtc tcaataatgt ctctagctcg 8820 ttggagtcta ctgactcaaa catttataaa gtgtcctaga aaggcctgac tcccctacaa 8880 ggctgtgtga tccttcaaac tcacatatgt gagccaataa aaccttgaga ctctag 8936 27 2431 PRT Rattus norvegicus 27 Met Asp Pro Gln Leu Arg Leu Leu Leu Glu Val Ser Tyr Glu Ala Ile 1 5 10 15 Val Asp Gly Gly Ile Asn Pro Ala Ser Leu Arg Gly Thr Asn Thr Gly 20 25 30 Val Trp Val Gly Val Ser Gly Ser Glu Ala Ser Glu Ala Leu Ser Arg 35 40 45 Asp Pro Glu Thr Leu Leu Gly Tyr Ser Met Val Gly Cys Gln Arg Ala 50 55 60 Met Met Ala Asn Arg Leu Ser Phe Phe Phe Asp Phe Lys Gly Pro Ser 65 70 75 80 Ile Ala Leu Asp Thr Ala Cys Ser Ser Ser Leu Leu Ala Leu Gln Asn 85 90 95 Ala Tyr Gln Ala Ile Arg Ser Gly Glu Cys Pro Ala Ala Ile Val Gly 100 105 110 Gly Ile Asn Leu Leu Leu Lys Pro Asn Thr Ser Val Gln Phe Met Lys 115 120 125 Leu Gly Met Leu Ser Pro Asp Gly Thr Cys Arg Ser Phe Asp Asp Ser 130 135 140 Gly Asn Gly Tyr Cys Arg Ala Glu Ala Val Val Ala Val Leu Leu Thr 145 150 155 160 Lys Lys Ser Leu Ala Arg Arg Val Tyr Ala Thr Ile Leu Asn Ala Gly 165 170 175 Thr Asn Thr Asp Gly Cys Lys Glu Gln Gly Val Thr Phe Pro Ser Gly 180 185 190 Glu Ala Gln Glu Gln Leu Ile Arg Ser Leu Tyr Gln Pro Gly Gly Val 195 200 205 Ala Pro Glu Ser Leu Glu Tyr Ile Glu Ala His Gly Thr Gly Thr Lys 210 215 220 Val Gly Asp Pro Gln Glu Leu Asn Gly Ile Thr Arg Ser Leu Cys Ala 225 230 235 240 Phe Arg Gln Ser Pro Leu Leu Ile Gly Ser Thr Lys Ser Asn Met Gly 245 250 255 His Pro Glu Pro Ala Ser Gly Leu Ala Ala Leu Thr Lys Val Leu Leu 260 265 270 Ser Leu Glu Asn Gly Val Trp Ala Pro Asn Leu His Phe His Asn Pro 275 280 285 Asn Pro Glu Ile Pro Ala Leu Leu Asp Gly Arg Leu Gln Val Val Asp 290 295 300 Arg Pro Leu Pro Val Arg Gly Gly Ile Val Gly Ile Asn Ser Phe Gly 305 310 315 320 Phe Gly Gly Ala Asn Val His Val Ile Leu Gln Pro Asn Thr Gln Gln 325 330 335 Ala Pro Ala Pro Ala Pro His Ala Ala Leu Pro His Leu Leu His Ala 340 345 350 Ser Gly Arg Thr Met Glu Ala Val Gln Gly Leu Leu Glu Gln Gly Arg 355 360 365 Gln His Ser Gln Asp Leu Ala Phe Val Ser Met Leu Asn Asp Ile Ala 370 375 380 Ala Thr Pro Thr Ala Ala Met Pro Phe Arg Gly Tyr Thr Val Leu Gly 385 390 395 400 Val Glu Gly His Val Gln Glu Val Gln Gln Val Pro Ala Ser Gln Arg 405 410 415 Pro Leu Trp Phe Ile Cys Ser Gly Met Gly Thr Gln Trp Arg Gly Met 420 425 430 Gly Leu Ser Leu Met Arg Leu Asp Ser Phe Arg Glu Ser Ile Leu Arg 435 440 445 Ser Asp Glu Ala Leu Lys Pro Leu Gly Val Lys Val Ser Asp Leu Leu 450 455 460 Leu Ser Thr Asp Glu His Thr Phe Asp Asp Ile Val His Ser Phe Val 465 470 475 480 Ser Leu Thr Ala Ile Gln Ile Ala Leu Ile Asp Leu Leu Thr Ser Met 485 490 495 Gly Leu Lys Pro Asp Gly Ile Ile Gly His Ser Leu Gly Glu Val Ala 500 505 510 Cys Gly Tyr Ala Asp Gly Cys Leu Ser Gln Arg Glu Ala Val Leu Ala 515 520 525 Ala Tyr Trp Arg Gly Gln Cys Ile Lys Asp Ala Asn Leu Pro Ala Gly 530 535 540 Ser Met Ala Ala Val Gly Leu Ser Trp Glu Glu Cys Lys Gln Arg Cys 545 550 555 560 Pro Pro Gly Val Val Pro Ala Cys His Asn Ser Glu Asp Thr Val Thr 565 570 575 Ile Ser Gly Pro Gln Ala Ala Val Asn Glu Phe Val Glu Gln Leu Lys 580 585 590 Gln Glu Gly Val Phe Ala Lys Glu Val Arg Thr Gly Gly Leu Ala Phe 595 600 605 His Ser Tyr Phe Met Glu Gly Ile Ala Pro Thr Leu Leu Gln Ala Leu 610 615 620 Lys Lys Val Ile Arg Glu Pro Arg Pro Arg Ser Ala Arg Trp Leu Ser 625 630 635 640 Thr Ser Ile Pro Glu Ala Gln Trp Gln Ser Ser Leu Ala Arg Thr Ser 645 650 655 Ser Ala Glu Tyr Asn Val Asn Asn Leu Val Ser Pro Val Leu Phe Gln 660 665 670 Glu Ala Leu Trp His Val Pro Glu His Ala Val Val Leu Glu Ile Ala 675 680 685 Pro His Ala Leu Leu Gln Ala Val Leu Lys Arg Gly Val Lys Pro Ser 690 695 700 Cys Thr Ile Ile Pro Leu Met Lys Arg Asp His Lys Asp Asn Leu Glu 705 710 715 720 Phe Phe Leu Thr Asn Leu Gly Lys Val His Leu Thr Gly Ile Asp Ile 725 730 735 Asn Pro Asn Ala Leu Phe Pro Pro Val Glu Phe Pro Val Pro Arg Gly 740 745 750 Thr Pro Leu Ile Ser Pro His Ile Lys Trp Asp His Ser Gln Thr Trp 755 760 765 Asp Ile Pro Val Ala Glu Asp Phe Pro Asn Gly Ser Ser Ser Ser Ser 770 775 780 Ala Thr Val Tyr Asn Ile Asp Ala Ser Ser Glu Ser Pro Asp His Tyr 785 790 795 800 Leu Val Asp His Cys Ile Asp Gly Arg Val Leu Phe Pro Gly Thr Gly 805 810 815 Tyr Leu Tyr Leu Val Trp Lys Thr Leu Ala Arg Ser Leu Ser Leu Ser 820 825 830 Leu Glu Glu Thr Pro Val Val Phe Glu Asn Val Thr Phe His Gln Ala 835 840 845 Thr Ile Leu Pro Arg Thr Gly Thr Val Pro Leu Glu Val Arg Leu Leu 850 855 860 Glu Ala Ser His Ala Phe Glu Val Ser Asp Ser Gly Asn Leu Ile Val 865 870 875 880 Ser Gly Lys Val Tyr Gln Trp Glu Asp Pro Asp Ser Lys Leu Phe Asp 885 890 895 His Pro Glu Val Pro Ile Pro Ala Glu Ser Glu Ser Val Ser Arg Leu 900 905 910 Thr Gln Gly Glu Val Tyr Lys Glu Leu Arg Leu Arg Gly Tyr Asp Tyr 915 920 925 Gly Pro His Phe Gln Gly Val Tyr Glu Ala Thr Leu Glu Gly Glu Gln 930 935 940 Gly Lys Leu Leu Trp Lys Asp Asn Trp Val Thr Phe Met Asp Thr Met 945 950 955 960 Leu Gln Ile Ser Ile Leu Gly Phe Ser Lys Gln Ser Leu Gln Leu Pro 965 970 975 Thr Arg Val Thr Ala Ile Tyr Ile Asp Pro Ala Thr His Leu Gln Lys 980 985 990 Val Tyr Met Leu Glu Gly Asp Thr Gln Val Ala Asp Val Thr Thr Ser 995 1000 1005 Arg Cys Leu Gly Val Thr Val Ser Gly Gly Val Tyr Ile Ser Arg 1010 1015 1020 Leu Gln Thr Thr Ala Thr Ser Arg Arg Gln Gln Glu Gln Leu Val 1025 1030 1035 Pro Thr Leu Glu Lys Phe Val Phe Thr Pro His Val Glu Pro Glu 1040 1045 1050 Cys Leu Ser Glu Ser Ala Ile Leu Gln Lys Glu Leu Gln Leu Cys 1055 1060 1065 Lys Gly Leu Ala Lys Ala Leu Gln Thr Lys Ala Thr Gln Gln Gly 1070 1075 1080 Leu Lys Met Thr Val Pro Gly Leu Glu Asp Leu Pro Gln His Gly 1085 1090 1095 Leu Pro Arg Leu Leu Ala Ala Ala Cys Gln Leu Gln Leu Asn Gly 1100 1105 1110 Asn Leu Gln Leu Glu Leu Gly Glu Val Leu Ala Arg Glu Arg Leu 1115 1120 1125 Leu Leu Pro Glu Asp Pro Leu Ile Ser Gly Leu Leu Asn Ser Gln 1130 1135 1140 Ala Leu Lys Ala Cys Ile Asp Thr Ala Leu Glu Asn Leu Ser Thr 1145 1150 1155 Leu Lys Met Lys Val Val Glu Val Leu Ala Gly Glu Gly His Leu 1160 1165 1170 Tyr Ser His Ile Ser Ala Leu Leu Asn Thr Gln Pro Met Leu Gln 1175 1180 1185 Leu Glu Tyr Thr Ala Thr Asp Arg His Pro Gln Ala Leu Lys Asp 1190 1195 1200 Val Gln Thr Lys Leu Gln Gln His Asp Val Ala Gln Gly Gln Trp 1205 1210 1215 Asp Pro Ser Gly Pro Ala Pro Thr Asn Leu Gly Ala Leu Asp Leu 1220 1225 1230 Val Val Cys Asn Cys Ala Leu Ala Thr Leu Gly Asp Pro Ala Leu 1235 1240 1245 Ala Leu Asp Asn Met Val Ala Ala Leu Lys Asp Gly Gly Phe Leu 1250 1255 1260 Leu Met His Thr Val Leu Lys Gly His Ala Leu Gly Glu Thr Leu 1265 1270 1275 Ala Cys Leu Pro Ser Glu Val Gln Pro Gly Pro Ser Phe Leu Ser 1280 1285 1290 Gln Glu Glu Trp Glu Ser Leu Phe Ser Arg Lys Ala Leu His Leu 1295 1300 1305 Val Gly Leu Lys Lys Ser Phe Tyr Gly Thr Ala Leu Phe Leu Cys 1310 1315 1320 Arg Arg Leu Ser Pro Gln Asp Lys Pro Ile Phe Leu Pro Val Glu 1325 1330 1335 Asp Thr Ser Phe Gln Trp Val Asp Ser Leu Lys Ser Ile Leu Ala 1340 1345 1350 Thr Ser Ser Ser Gln Pro Val Trp Leu Thr Ala Met Asn Cys Pro 1355 1360 1365 Thr Ser Gly Val Val Gly Leu Val Asn Cys Leu Arg Lys Glu Pro 1370 1375 1380 Gly Gly His Arg Ile Arg Cys Ile Leu Leu Ser Asn Leu Ser Ser 1385 1390 1395 Thr Ser His Val Pro Lys Leu Asp Pro Gly Ser Ser Glu Leu Gln 1400 1405 1410 Lys Val Leu Glu Ser Asp Leu Val Met Asn Val Tyr Arg Asp Gly 1415 1420 1425 Ala Trp Gly Ala Phe Arg His Phe Gln Leu Glu Gln Asp Lys Pro 1430 1435 1440 Glu Glu Gln Thr Ala His Ala Phe Val Asn Val Leu Thr Arg Gly 1445 1450 1455 Asp Leu Ala Ser Ile Arg Trp Val Ser Ser Pro Leu Lys His Met 1460 1465 1470 Gln Pro Pro Ser Ser Ser Gly Ala Gln Leu Cys Thr Val Tyr Tyr 1475 1480 1485 Ala Ser Leu Asn Phe Arg Asp Ile Met Leu Ala Thr Gly Lys Leu 1490 1495 1500 Ser Pro Asp Ala Ile Pro Gly Lys Trp Ala Ser Arg Asp Cys Met 1505 1510 1515 Leu Gly Met Glu Phe Ser Gly Arg Asp Lys Cys Gly Arg Arg Val 1520 1525 1530 Met Gly Leu Val Pro Ala Glu Gly Leu Ala Thr Ser Val Leu Leu 1535 1540 1545 Ser Pro Asp Phe Leu Trp Asp Val Pro Ser Ser Trp Thr Leu Glu 1550 1555 1560 Glu Ala Ala Ser Val Pro Val Val Tyr Thr Thr Ala Tyr Tyr Ser 1565 1570 1575 Leu Val Val Arg Gly Arg Ile Gln His Gly Glu Thr Val Leu Ile 1580 1585 1590 His Ser Gly Ser Gly Gly Val Gly Gln Ala Ala Ile Ser Ile Ala 1595 1600 1605 Leu Ser Leu Gly Cys Arg Val Phe Thr Thr Val Gly Ser Ala Glu 1610 1615 1620 Lys Arg Ala Tyr Leu Gln Ala Arg Phe Pro Gln Leu Asp Asp Thr 1625 1630 1635 Ser Phe Ala Asn Ser Arg Asp Thr Ser Phe Glu Gln His Val Leu 1640 1645 1650 Leu His Thr Gly Gly Lys Gly Val Asp Leu Val Leu Asn Ser Leu 1655 1660 1665 Ala Glu Glu Lys Leu Gln Ala Ser Val Arg Cys Leu Ala Gln His 1670 1675 1680 Gly Arg Phe Leu Glu Ile Gly Lys Phe Asp Leu Ser Asn Asn His 1685 1690 1695 Pro Leu Gly Met Ala Ile Phe Leu Lys Asn Val Thr Phe His Gly 1700 1705 1710 Ile Leu Leu Asp Ala Leu Phe Glu Gly Ala Asn Asp Ser Trp Arg 1715 1720 1725 Glu Val Ala Glu Leu Leu Lys Ala Gly Ile Arg Asp Gly Val Val 1730 1735 1740 Lys Pro Leu Lys Cys Thr Val Phe Pro Lys Ala Gln Val Glu Asp 1745 1750 1755 Ala Phe Arg Tyr Met Ala Gln Gly Lys His Ile Gly Lys Val Leu 1760 1765 1770 Val Gln Val Arg Glu Glu Glu Pro Glu Ala Met Leu Pro Gly Ala 1775 1780 1785 Gln Pro Thr Leu Ile Ser Ala Ile Ser Lys Thr Phe Cys Pro Glu 1790 1795 1800 His Lys Ser Tyr Ile Ile Thr Gly Gly Leu Gly Gly Phe Gly Leu 1805 1810 1815 Glu Leu Ala Arg Trp Leu Val Leu Arg Gly Ala Gln Arg Leu Val 1820 1825 1830 Leu Thr Ser Arg Ser Gly Ile Arg Thr Gly Tyr Gln Ala Lys His 1835 1840 1845 Val Arg Glu Trp Arg Arg Gln Gly Ile His Val Leu Val Ser Thr 1850 1855 1860 Ser Asn Val Ser Ser Leu Glu Gly Ala Arg Ala Leu Ile Ala Glu 1865 1870 1875 Ala Thr Lys Leu Gly Pro Val Gly Gly Val Phe Asn Leu Ala Met 1880 1885 1890 Val Leu Arg Asp Ala Met Leu Glu Asn Gln Thr Pro Glu Leu Phe 1895 1900 1905 Gln Asp Val Asn Lys Pro Lys Tyr Asn Gly Thr Leu Asn Leu Asp 1910 1915 1920 Arg Ala Thr Arg Glu Ala Cys Pro Glu Leu Asp Tyr Phe Val Ala 1925 1930 1935 Phe Ser Ser Val Ser Cys Gly Arg Gly Asn Ala Gly Gln Ser Asn 1940 1945 1950 Tyr Gly Phe Ala Asn Ser Thr Met Glu Arg Ile Cys Glu Gln Arg 1955 1960 1965 Arg His Asp Gly Leu Pro Gly Leu Ala Val Gln Trp Gly Ala Ile 1970 1975 1980 Gly Asp Val Gly Ile Ile Leu Glu Ala Met Gly Thr Asn Asp Thr 1985 1990 1995 Val Val Gly Gly Thr Leu Pro Gln Arg Ile Ser Ser Cys Met Glu 2000 2005 2010 Val Leu Asp Leu Phe Leu Asn Gln Pro His Ala Val Leu Ser Ser 2015 2020 2025 Phe Val Leu Ala Glu Lys Lys Ala Val Ala His Gly Asp Gly Glu 2030 2035 2040 Ala Gln Arg Asp Leu Val Lys Ala Val Ala His Ile Leu Gly Ile 2045 2050 2055 Arg Asp Leu Ala Gly Ile Asn Leu Asp Ser Ser Leu Ala Asp Leu 2060 2065 2070 Gly Leu Asp Ser Leu Met Gly Val Glu Val Arg Gln Ile Leu Glu 2075 2080 2085 Arg Glu His Asp Leu Val Leu Pro Ile Arg Glu Val Arg Gln Leu 2090 2095 2100 Thr Leu Arg Lys Leu Gln Glu Met Ser Ser Lys Ala Gly Ser Asp 2105 2110 2115 Thr Glu Leu Ala Ala Pro Lys Ser Lys Asn Asp Thr Ser Leu Lys 2120 2125 2130 Gln Ala Gln Leu Asn Leu Ser Ile Leu Leu Val Asn Pro Glu Gly 2135 2140 2145 Pro Thr Leu Thr Arg Leu Asn Ser Val Gln Ser Ser Glu Arg Pro 2150 2155 2160 Leu Phe Leu Val His Pro Ile Glu Gly Ser Ile Thr Val Phe His 2165 2170 2175 Ser Leu Ala Ala Lys Leu Ser Val Pro Thr Tyr Gly Leu Gln Cys 2180 2185 2190 Thr Gln Ala Ala Pro Leu Asp Ser Ile Pro Asn Leu Ala Ala Tyr 2195 2200 2205 Tyr Ile Asp Cys Ile Lys Gln Val Gln Pro Glu Gly Pro Tyr Arg 2210 2215 2220 Val Ala Gly Tyr Ser Phe Gly Ala Cys Val Ala Phe Glu Met Cys 2225 2230 2235 Ser Gln Leu Gln Ala Gln Gln Gly Pro Ala Pro Ala His Asn Asn 2240 2245 2250 Leu Phe Leu Phe Asp Gly Ser His Thr Tyr Val Leu Ala Tyr Thr 2255 2260 2265 Gln Ser Tyr Arg Ala Lys Leu Thr Pro Gly Cys Glu Ala Glu Ala 2270 2275 2280 Glu Ala Glu Ala Ile Cys Phe Phe Ile Lys Gln Phe Val Asp Ala 2285 2290 2295 Glu His Ser Lys Val Leu Glu Ala Leu Leu Pro Leu Lys Ser Leu 2300 2305 2310 Glu Asp Arg Val Ala Ala Ala Val Asp Leu Ile Thr Arg Ser His 2315 2320 2325 Gln Ser Leu Asp Arg Arg Asp Leu Ser Phe Ala Ala Val Ser Phe 2330 2335 2340 Tyr Tyr Lys Leu Arg Ala Ala Asp Gln Tyr Lys Pro Lys Ala Lys 2345 2350 2355 Tyr His Gly Asn Val Ile Leu Leu Arg Ala Lys Thr Gly Gly Thr 2360 2365 2370 Tyr Gly Glu Asp Leu Gly Ala Asp Tyr Asn Leu Ser Gln Val Cys 2375 2380 2385 Asp Gly Lys Val Ser Val His Ile Ile Glu Gly Asp His Arg Thr 2390 2395 2400 Leu Leu Glu Gly Arg Gly Leu Glu Ser Ile Ile Asn Ile Ile His 2405 2410 2415 Ser Ser Leu Ala Glu Pro Arg Val Ser Val Arg Glu Gly 2420 2425 2430 28 9345 DNA Gallus gallus 28 agaacctgct caatggggtt gatatggtca cagaggacga tcggaggtgg aagccaggga 60 tttatggact gcccaaaaga aatggaaagc tcaaggacat aaaaaaattc gatgcctcct 120 tctttgggtc caccccaaac aagctcatac aatggatcct ccagttcgct tgttgttgga 180 agtttcttat gaggctattt tggatggagg cattaatcca actgccctcc gtggcacaga 240 cacgggtgta tgggttggtg caagtggctc agaagctgct gaagccctta gccaagatcc 300 agaagagctt ttgggataca gtatgactgg ctgccagcgt gctatgcttg ccaacaggat 360 ttcttacttc tatgatttta caggaccaag cttaactatc gacacagcct gctcctccag 420 tctcatggct ttagaaaatg cttataaagc aattcgtcac ggacagtgca gtgcagccct 480 ggtaggaggg gtcaacattc tgctgaagcc caacacttct gtgcagttca tgaagctggg 540 catgcttagt cctgatggtg cctgcaaggc tttcgatgtt tcaggaaatg ggtattgtcg 600 ctctgaagct gttgttgttg tgctcttgac caagaaatcc atggctaaac gcgtctatgc 660 cactatagtc aatgctggga gtaacactga tggctttaag gagcaaggtg tgacattccc 720 atctggagag atgcagcagc agctggttgg ttctctgtac agagaatgtg gtatcaagcc 780 tggagatgtg gagtatgttg aagctcatgg gacaggcacc aaggttggag atcctcaaga 840 agtaaatggc attgtaaatg tcttctgcca gtgtgagaga gagcctctgt taattggatc 900 aaccaagtca aacatgggtc atccagagcc tgcttctggg cttgctgcat tagccaaggt 960 cattctttct ctggaacatg gactgtgggc tccaaatctt catttcaatg atccaaatcc 1020 agatattcct gctttacacg atggctcctt gaaggtggtt tgcaaaccaa caccggtgaa 1080 aggtggcctt gtcagcatca attcttttgg ctttggaggc tctaatgctc atgttattct 1140 gaggccaaat gagaagaaat gtcagcctca agagacttgt aacttgccaa gactggttca 1200 agtttgtggc agaacacagg aagctgtgga aatactaatt gaagaaagca ggaaacatgg 1260 aggatgcagt ccatttttaa gcctgctcag tgatatctct gcagttcctg tatcttctat 1320 gccctacagg ggctacacac tagttggcac tgagagtgac ataacagaga ttcagcaagt 1380 tcaagcatct ggtagaccac tctggtacat ctgctcaggc atgggaacac agtggaaagg 1440 tatgggcctg agccttatga aattggatct gtttcgccag tctatattgc gctcagatga 1500 ggctttgaag agcacaggac tgaaggtctc agacctgctt ctgaatgcag atgagaacac 1560 ttttgatgac actgtccatg cttttgttgg actagctgct atacagattg cccaaattga 1620 tgtgctaaag gctgcgggtc tgcaacctga tgggattttg ggccactcag tgggagaact 1680 agcttgtggc tatgcagata attccttaag tcatgaagaa gctgttcttg ctgcttattg 1740 gaggggccga tgtgtgaaag aggccaaatt gcccccggga gggatggctg ctgttggtct 1800 gacatgggag gaatgtaagc agcgctgtcc tccaaacgtg gtaccagcat gtcacaactc 1860 tgaggatact gtcactgttt cggggcctct ggattctgtg tctgagtttg taaccaaact 1920 gaagaaagat ggggtgtttg caaaggaggt gcgcagcgcc ggagttgcat ttcattccta 1980 ttacatggca tccattgcac cagcactgct cagtgcactg aaaaaggtca ttccacaccc 2040 taagcctcgt tcagcacggt ggatcagtac atctatccct gaatctcagt ggcagagtga 2100 tcttgctagg aattcctctg cagagtatca tgtgaacaac ctagtgaatc ctgtgctgtt 2160 ccatgaaggc ctgaagcata ttccagagaa tgctgttgta gtggagattg ctccacatgc 2220 tctcttacag gctatcttga ggagaacttt gaagccaact tgcactattc tacctctgat 2280 gaagaaggac cacaaaaata acttggagtt cttcctaacg cagactggaa agattcattt 2340 aactgggata aatgttcttg gaaataactt gttcccacct gtggaatacc ctgtccctgt 2400 gggaacacct ctcatttctc catatatcaa atgggaccac agccaagact gggatgttcc 2460 aaaagctgaa gacttcccct caggttccaa aggctctgcg tctgcttcag tctacaacat 2520 cgatgtgagt cctgactctc ctgaccatta cttggttggc cattgcattg atggcagagt 2580 cctgtaccca gcaactgggt acttagtgct ggcgtggcga actctggcac gatctcttgg 2640 catggtcatg gaacaaacag ctgttatgtt tgaagaagtt acaatccatc aggcaactat 2700 ccttcccaaa aagggatcaa cacagctgga agtacgaatc atgcctgctt ctcacagctt 2760 tgaagtgtca gggaatggga atttggctgt gagtgggaag atctccctcc tagaaaacga 2820 tgctctgaag aactttcata accagctggc tgactttcag agtcaagcaa acgtgactgc 2880 gaagtctggc ctcttgatgg aagatgttta ccaagagctg catcttcgtg gatataacta 2940 tggaccaact tttcagggtg ttctggaatg caacagtgaa ggaagtgcag ggaaaattct 3000 gtggaatgga aactgggtaa ccttccttga caccctgcta cacttgatag tcttagcaga 3060 gactgggcgc agtctacgat tgcccaccag gattcgctca gtgtatattg accctgtgct 3120 tcatcaggag caggtgtacc agtaccagga caatgtagaa gcttttgatg ttgttgttga 3180 ccgctgtctt gatagcctca aagcaggagg tgttcagatc aatggacttc atgcctcggt 3240 ggcaccacgg cgacaacagg agcggatctc tcccactctg gaaaaattct cctttgttcc 3300 ctatattgag agtgactgtt tgtcttccag tacccagctt catgcctacc tggagcactg 3360 caaaggcctg atccagaaat tacaagctaa gatggcattg cacggagtca aactagttat 3420 ccatggccta gaaaccaacg gggctgctgc aggatcccca cccacacaga agggccttca 3480 gcatatcctt actgaaatct gccatctgga actgaatgga aacctacatt ctgagctgga 3540 acagattgtg actcaggaga agatgcacct ccaggacgat ccccttctca atggcttgct 3600 ggattcttca gagttgaaga cttgcctgga tgtggcaaag gagaacacga ccagtcacag 3660 gatgaagata gtggaggctc tggcaggaag tggacgtctg ttctctcgtg tccaaagtat 3720 tctgaatact cagcccctgt tgcagctgga ctacattgcc actgactgca cccctgaaac 3780 tctttcaaat gatgaaacag agctgcacga tgctggaatc tcctttagcc agtgggatcc 3840 ctctagcctt ccctctggaa atctgaccaa tgctgacctg gcagtatgca actgttcaac 3900 aagtgttctg gggaacacag ctgaaattat ctctaactta gcagctgcag tgaaagaagg 3960 agggtttgtt ttgctgcaca cccttcttaa agaggaaact cttggagaaa ttgtcagctt 4020 tcttacaagt ccagacctac agcaagagca cagcttcctg tctcaggcac agtgggagga 4080 gttattcagc aaggcctcat tgaatctggt tgcaatgaag agatctttct ttggctcagt 4140 tattttcctg tgtcgacggc agtcccctgc caaagcaccc attcttctgc cagtagatga 4200 cactcattat aagtgggttg actccttaaa ggagatcttg gctgactcat cagagcagcc 4260 tctgtggttg actgccacca attgtgggaa ctctggaatt ttgggtatgg tgaactgcct 4320 ccgcctggaa gcagagggcc acagaatcag gtgtgtgttt gtttccaacc tgagcccttc 4380 atcaactgtc ccagccacta gtctttcttc cctggagatg cagaagatta ttgagagaga 4440 tctggtgatg aatgtgtatc gtgatggaaa gtggggttcc ttcaggcatc tcccattgca 4500 gcaagctcag cctcaggagc tgacagaata tgcctacgta aatgtgttga ctcgtggaga 4560 tctctcttcc cttcgttgga ttgtttcccc acttcgacac ttccaaacaa ccaatccaaa 4620 tgttcagctc tgcaaagtct actatgcatc tctcaatttc cgggacatta tgctggcaac 4680 aggaaagctt tctccagatg ctatccctgg taactggacg ttgcagcagt gcatgctggg 4740 catggagttc tcaggacggg acctggctgg aaggagagtg atgggattgc tgccagcaaa 4800 agggctggcg acagtggtgg actgtgacaa gaggtttcta tgggaagtgc ctgaaaactg 4860 gactctggaa gaagcagctt cggtgcctgt ggtttatgcc actgcttatt atgctttggt 4920 ggttcgaggt ggtatgaaga agggggagag tgtcctcatt cactctggct caggaggtgt 4980 gggcgcaagc agccattgcc atcgccttga gcatgggctg gcgcgtgttt ttgctactgt 5040 aggctctgct gagaaacgtg agtatctcca agcaaggttc ccacagctgg atgctaatag 5100 ctttgccagc tcccgaaata caacctttga gcaacacata ctgcgagtta ccaatgggaa 5160 aggtgtcaac cttgtgttaa attccttggc agaagagaag ctccaagcca gtttgcgttg 5220 tcttgctcaa catgggcgct tcttggaaat aggcaaattt gatctatcaa acaacagcca 5280 gcttggaatg gctcttttcc tcaagaatgt ggcgtttcat ggaatcctgc tggattcaat 5340 ctttgaggaa ggaaaccaag agtgggaggt ggtatcagag ttgttgacaa aaggcataaa 5400 agatggtgtg gtaaagcccc tgagaaccac agtcttcggt aaagaagagg tagaagctgc 5460 cttcaggttc atggcgcaag gaaaacatat tggcaaagtt atgatcaaga tccaagaaga 5520 ggagaagcaa tatcctttaa ggtctgaacc agtaaaactc tctgccatct cccgaacttc 5580 ctgcccacct accaagtctt acatcatcac agggggccta ggaggatttg ggcttgagtt 5640 ggcacagtgg ctaattgaga gaggagcaca gaagcttgta ctgacatctc gatctggcat 5700 acgaactggc taccaggcta aatgtgttag agaatggaag gcgctgggaa tccaagtgtt 5760 ggtctctacc agtgatgttg gaactctaga aggaacgcag cttttgatag aagaggcttt 5820 gaagctcgga ccagttgggg gcatctttaa tttggctgtg gtccttaaag atgccatgat 5880 tgaaaatcag accccggaat tattctggga ggtcaacaag cccaagtatt caggcaccct 5940 tcatttggac tgggtgactc gtaagaagtg cccagacctg gactattttg ttgtattctc 6000 ctctgtaagc tgtggaagag gaaatgctgg gcaaagtaat tatggctttg ctaattctgc 6060 catggagcgt atctgtgagc agcggcatca cgatgggctc ccaggcctgg cagtccagtg 6120 gggagccatt ggtgatgtgg gcatcctgaa ggcaatggga aacagggagg ttgtgattgg 6180 gggaaccgtt ctccagcaaa tcagctcctg cctggaggtg ctcgatatgt tcctgaatca 6240 acctcatcct gttatgtcca gttttgtcct agcagagaag gtctctgtga aaagtgaagg 6300 aggaagtcaa cgggatcttg tagaagctgt tgctcatatc cttggtgttc gtgacgtgag 6360 cagtctgaat gctgagagct ccctagcaga cttgggcctg gattccttga tgggtgtgga 6420 ggtgcgccag acgctggaga gagactacga catcgtaatg accatgaggg agatccgact 6480 cctcaccatc aacaaactgc gtgaactgtc ctccaagact gggacagcag aggagctgaa 6540 gccatcacaa gtgttgaaga caggcccagg tgagcctcca aaactggatt tgaacaactt 6600 gctggtgaat ccagaagggc caacgattac ccgtctcaat gaagttcaga gcacagaacg 6660 ccctcttttc cttgttcacc ccattgaggg atccattgca gtcttctata ctcttgcctc 6720 caaacttcat atgccctgct atggactcca gtgcacaaaa gctgctccct tggacagcat 6780 acagagcctg gcatcctatt atattgactg tatgaagcag atacagcctg aaggacctta 6840 tcgcattgct ggatactctt ttggtgcctg cgtagccttt gaaatgtgct cccagctgca 6900 agcacaacaa aatgcttccc atgcactcaa cagtttattc ctctttgatg ggtctcattc 6960 ctttgtggca gcatacactc agtgtttttc cttttctctt tttcagagct acagagcaaa 7020 gctgacccaa ggaaatgagg ctgcgttgga gacagaagca ctgtgtgcct ttgttcagca 7080 gtttacaggc attgaataca ataagttgtt ggagattctt ctgcccttgg aagatctgga 7140 ggctcgtgtc aatgctgctg cagaccttat aactcagatt cataaaaaca tcaaccgtga 7200 agcactcagc tttgctgctg cttcctttta ccataagctg aaggctgctg acaagtatat 7260 accagaatcc aagtatcatg ggaacgtgac actgatgcgg gcaaagactc acaatgagta 7320 tgaagaaggt ctgggtggag actacagact ctcagaggtc tgcgatggaa aagtatcagt 7380 ccacatcatt gaaggagatc accgcacctt attggaggga gatggtgttg aatcaatcat 7440 tgggatcatc catggctcac tggcagagcc acgtgtcagt gtcagagaag gttaacttct 7500 gccacttact gtcagtggtg aagaaaatgc caacaacatt cctagttatg acagacccca 7560 aggaactctt cctgttgaac aacatctcat ctctctgctg ccagagctgg gaaggccagc 7620 tgaacttgat tggtctcttt gtttcctctc tcactcagtc atctttccta actttcacgt 7680 gttctctctc tctcctcttc ccttcctatg ctttgtctat ttccccacta tccctgcccg 7740 tgttactgcg gtgctgtgac tgtcactgtg caccgggggt tccccggcga tggtggcttc 7800 ccacagcttt ggcagtatgt ttttcaaatt taggagtaga cttctacgtg ctctatattg 7860 ttttgtctta acagtattcc aaagggtaag tgatagcact tgttgaccaa gcccagtgag 7920 cagagagggg aactgcagct gatttcggag atacctgttg tctgtgaaga atctgtctgt 7980 agtgaggtca gaaagagaat tccatttgag gcttttgtaa ctatattttt ttaatttgat 8040 atagtctaag tatttattgt gtcaaatcag agacttcttg ctttgtttta atttatcgtg 8100 ggtatcagaa aaggaaacat ccgttttgaa gggataggtt cattctacaa ggggaggttg 8160 cccatttgtt aaaccaaagt gcatctatgg aacagcccat ttcttttttt tttttaagtt 8220 gattttttgt ttgtgtttcg ttttttgttg tttgtttttt gtggcgtttt gttaattttg 8280 attagtgatt tttctgtgtg tggtttttct ttcccccccc cccccaccct gccttgttca 8340 gaagggtgga agtgaggttc cttgccatca cccacccttg tggggagaga ggcgtggagg 8400 gcaggatgga tggttcaaca gatgccactg tattgaacag ccttaacttg ggctgataca 8460 agcaggcaga gctctcccta ggtatgtact tagtttatat ctctgcaagg ttctgtgctt 8520 tgcattacca gaaacacagt aaagcattac ggctattgct tcacctttgt tccttcccac 8580 ctccagttgc tccatccaac caggcatttg gaatgtcagg gggaatagag ttctccattg 8640 gtcacggtat aaatcctcct acccttgctc tcccataacc aaagttcatg caaacataga 8700 aggcatctac ccagtacccc agtgtatttt atgtagcata ggcttgctta agccttgagt 8760 atgcattttc ctctggcagt gagactggag atcccacata agttagctaa gtaaaagttt 8820 gatggcatga ttttaagata cagtaccttt ttaaaggaaa cttgcataaa attcaattta 8880 aaaatgactg acttttgcta tgctggatct gtcttttcca aaatcagtaa atcctcttga 8940 cgcctatgat acagaggaga cctgaatagc aatgaagtac caaccaggag gcattccact 9000 gcctctcaga acttctgtaa acccctgttc tttctgtatt catcccctag tgaagcatcc 9060 tgtgagttca ggagcattcc agtgagagga acagctggtt cctcgtggca ggttctacct 9120 agcgtctctt gcttatacaa ccctctgtgg agagtggctg ggttaactgg ttttagtttt 9180 ataaagtatt tcttttgtga aatctgaaat acaaacaaca taatgtcagc ttaaagcatt 9240 tctagaatta agttttgttt tttacttttt tttttttttt tttttaatct gaagagtgtc 9300 tttttcctct ttggctttcc tagaattaaa cagaattgat cactg 9345 29 2447 PRT Gallus gallus 29 Met Asp Pro Pro Val Arg Leu Leu Leu Glu Val Ser Tyr Glu Ala Ile 1 5 10 15 Leu Asp Gly Gly Ile Asn Pro Thr Ala Leu Arg Gly Thr Asp Thr Gly 20 25 30 Val Trp Val Gly Ala Ser Gly Ser Glu Ala Ala Glu Ala Leu Ser Gln 35 40 45 Asp Pro Glu Glu Leu Leu Gly Tyr Ser Met Thr Gly Cys Gln Arg Ala 50 55 60 Met Leu Ala Asn Arg Ile Ser Tyr Phe Tyr Asp Phe Thr Gly Pro Ser 65 70 75 80 Leu Thr Ile Asp Thr Ala Cys Ser Ser Ser Leu Met Ala Leu Glu Asn 85 90 95 Ala Tyr Lys Ala Ile Arg His Gly Gln Cys Ser Ala Ala Leu Val Gly 100 105 110 Gly Val Asn Ile Leu Leu Lys Pro Asn Thr Ser Val Gln Phe Met Lys 115 120 125 Leu Gly Met Leu Ser Pro Asp Gly Ala Cys Lys Ala Phe Asp Val Ser 130 135 140 Gly Asn Gly Tyr Cys Arg Ser Glu Ala Val Val Val Val Leu Leu Thr 145 150 155 160 Lys Lys Ser Met Ala Lys Arg Val Tyr Ala Thr Ile Val Asn Ala Gly 165 170 175 Ser Asn Thr Asp Gly Phe Lys Glu Gln Gly Val Thr Phe Pro Ser Gly 180 185 190 Glu Met Gln Gln Gln Leu Val Gly Ser Leu Tyr Arg Glu Cys Gly Ile 195 200 205 Lys Pro Gly Asp Val Glu Tyr Val Glu Ala His Gly Thr Gly Thr Lys 210 215 220 Val Gly Asp Pro Gln Glu Val Asn Gly Ile Val Asn Val Phe Cys Gln 225 230 235 240 Cys Glu Arg Glu Pro Leu Leu Ile Gly Ser Thr Lys Ser Asn Met Gly 245 250 255 His Pro Glu Pro Ala Ser Gly Leu Ala Ala Leu Ala Lys Val Ile Leu 260 265 270 Ser Leu Glu His Gly Leu Trp Ala Pro Asn Leu His Phe Asn Asp Pro 275 280 285 Asn Pro Asp Ile Pro Ala Leu His Asp Gly Ser Leu Lys Val Val Cys 290 295 300 Lys Pro Thr Pro Val Lys Gly Gly Leu Val Ser Ile Asn Ser Phe Gly 305 310 315 320 Phe Gly Gly Ser Asn Ala His Val Ile Leu Arg Pro Asn Glu Lys Lys 325 330 335 Cys Gln Pro Gln Glu Thr Cys Asn Leu Pro Arg Leu Val Gln Val Cys 340 345 350 Gly Arg Thr Gln Glu Ala Val Glu Ile Leu Ile Glu Glu Ser Arg Lys 355 360 365 His Gly Gly Cys Ser Pro Phe Leu Ser Leu Leu Ser Asp Ile Ser Ala 370 375 380 Val Pro Val Ser Ser Met Pro Tyr Arg Gly Tyr Thr Leu Val Gly Thr 385 390 395 400 Glu Ser Asp Ile Thr Glu Ile Gln Gln Val Gln Ala Ser Gly Arg Pro 405 410 415 Leu Trp Tyr Ile Cys Ser Gly Met Gly Thr Gln Trp Lys Gly Met Gly 420 425 430 Leu Ser Leu Met Lys Leu Asp Leu Phe Arg Gln Ser Ile Leu Arg Ser 435 440 445 Asp Glu Ala Leu Lys Ser Thr Gly Leu Lys Val Ser Asp Leu Leu Leu 450 455 460 Asn Ala Asp Glu Asn Thr Phe Asp Asp Thr Val His Ala Phe Val Gly 465 470 475 480 Leu Ala Ala Ile Gln Ile Ala Gln Ile Asp Val Leu Lys Ala Ala Gly 485 490 495 Leu Gln Pro Asp Gly Ile Leu Gly His Ser Val Gly Glu Leu Ala Cys 500 505 510 Gly Tyr Ala Asp Asn Ser Leu Ser His Glu Glu Ala Val Leu Ala Ala 515 520 525 Tyr Trp Arg Gly Arg Cys Val Lys Glu Ala Lys Leu Pro Pro Gly Gly 530 535 540 Met Ala Ala Val Gly Leu Thr Trp Glu Glu Cys Lys Gln Arg Cys Pro 545 550 555 560 Pro Asn Val Val Pro Ala Cys His Asn Ser Glu Asp Thr Val Thr Val 565 570 575 Ser Gly Pro Leu Asp Ser Val Ser Glu Phe Val Thr Lys Leu Lys Lys 580 585 590 Asp Gly Val Phe Ala Lys Glu Val Arg Ser Ala Gly Val Ala Phe His 595 600 605 Ser Tyr Tyr Met Ala Ser Ile Ala Pro Ala Leu Leu Ser Ala Leu Lys 610 615 620 Lys Val Ile Pro His Pro Lys Pro Arg Ser Ala Arg Trp Ile Ser Thr 625 630 635 640 Ser Ile Pro Glu Ser Gln Trp Gln Ser Asp Leu Ala Arg Asn Ser Ser 645 650 655 Ala Glu Tyr His Val Asn Asn Leu Val Asn Pro Val Leu Phe His Glu 660 665 670 Gly Leu Lys His Ile Pro Glu Asn Ala Val Val Val Glu Ile Ala Pro 675 680 685 His Ala Leu Leu Gln Ala Ile Leu Arg Arg Thr Leu Lys Pro Thr Cys 690 695 700 Thr Ile Leu Pro Leu Met Lys Lys Asp His Lys Asn Asn Leu Glu Phe 705 710 715 720 Phe Leu Thr Gln Thr Gly Lys Ile His Leu Thr Gly Ile Asn Val Leu 725 730 735 Gly Asn Asn Leu Phe Pro Pro Val Glu Tyr Pro Val Pro Val Gly Thr 740 745 750 Pro Leu Ile Ser Pro Tyr Ile Lys Trp Asp His Ser Gln Asp Trp Asp 755 760 765 Val Pro Lys Ala Glu Asp Phe Pro Ser Gly Ser Lys Gly Ser Ala Ser 770 775 780 Ala Ser Val Tyr Asn Ile Asp Val Ser Pro Asp Ser Pro Asp His Tyr 785 790 795 800 Leu Val Gly His Cys Ile Asp Gly Arg Val Leu Tyr Pro Ala Thr Gly 805 810 815 Tyr Leu Val Leu Ala Trp Arg Thr Leu Ala Arg Ser Leu Gly Met Val 820 825 830 Met Glu Gln Thr Ala Val Met Phe Glu Glu Val Thr Ile His Gln Ala 835 840 845 Thr Ile Leu Pro Lys Lys Gly Ser Thr Gln Leu Glu Val Arg Ile Met 850 855 860 Pro Ala Ser His Ser Phe Glu Val Ser Gly Asn Gly Asn Leu Ala Val 865 870 875 880 Ser Gly Lys Ile Ser Leu Leu Glu Asn Asp Ala Leu Lys Asn Phe His 885 890 895 Asn Gln Leu Ala Asp Phe Gln Ser Gln Ala Asn Val Thr Ala Lys Ser 900 905 910 Gly Leu Leu Met Glu Asp Val Tyr Gln Glu Leu His Leu Arg Gly Tyr 915 920 925 Asn Tyr Gly Pro Thr Phe Gln Gly Val Leu Glu Cys Asn Ser Glu Gly 930 935 940 Ser Ala Gly Lys Ile Leu Trp Asn Gly Asn Trp Val Thr Phe Leu Asp 945 950 955 960 Thr Leu Leu His Leu Ile Val Leu Ala Glu Thr Gly Arg Ser Leu Arg 965 970 975 Leu Pro Thr Arg Ile Arg Ser Val Tyr Ile Asp Pro Val Leu His Gln 980 985 990 Glu Gln Val Tyr Gln Tyr Gln Asp Asn Val Glu Ala Phe Asp Val Val 995 1000 1005 Val Asp Arg Cys Leu Asp Ser Leu Lys Ala Gly Gly Val Gln Ile 1010 1015 1020 Asn Gly Leu His Ala Ser Val Ala Pro Arg Arg Gln Gln Glu Arg 1025 1030 1035 Ile Ser Pro Thr Leu Glu Lys Phe Ser Phe Val Pro Tyr Ile Glu 1040 1045 1050 Ser Asp Cys Leu Ser Ser Ser Thr Gln Leu His Ala Tyr Leu Glu 1055 1060 1065 His Cys Lys Gly Leu Ile Gln Lys Leu Gln Ala Lys Met Ala Leu 1070 1075 1080 His Gly Val Lys Leu Val Ile His Gly Leu Glu Thr Asn Gly Ala 1085 1090 1095 Ala Ala Gly Ser Pro Pro Thr Gln Lys Gly Leu Gln His Ile Leu 1100 1105 1110 Thr Glu Ile Cys His Leu Glu Leu Asn Gly Asn Leu His Ser Glu 1115 1120 1125 Leu Glu Gln Ile Val Thr Gln Glu Lys Met His Leu Gln Asp Asp 1130 1135 1140 Pro Leu Leu Asn Gly Leu Leu Asp Ser Ser Glu Leu Lys Thr Cys 1145 1150 1155 Leu Asp Val Ala Lys Glu Asn Thr Thr Ser His Arg Met Lys Ile 1160 1165 1170 Val Glu Ala Leu Ala Gly Ser Gly Arg Leu Phe Ser Arg Val Gln 1175 1180 1185 Ser Ile Leu Asn Thr Gln Pro Leu Leu Gln Leu Asp Tyr Ile Ala 1190 1195 1200 Thr Asp Cys Thr Pro Glu Thr Leu Ser Asn Asp Glu Thr Glu Leu 1205 1210 1215 His Asp Ala Gly Ile Ser Phe Ser Gln Trp Asp Pro Ser Ser Leu 1220 1225 1230 Pro Ser Gly Asn Leu Thr Asn Ala Asp Leu Ala Val Cys Asn Cys 1235 1240 1245 Ser Thr Ser Val Leu Gly Asn Thr Ala Glu Ile Ile Ser Asn Leu 1250 1255 1260 Ala Ala Ala Val Lys Glu Gly Gly Phe Val Leu Leu His Thr Leu 1265 1270 1275 Leu Lys Glu Glu Thr Leu Gly Glu Ile Val Ser Phe Leu Thr Ser 1280 1285 1290 Pro Asp Leu Gln Gln Glu His Ser Phe Leu Ser Gln Ala Gln Trp 1295 1300 1305 Glu Glu Leu Phe Ser Lys Ala Ser Leu Asn Leu Val Ala Met Lys 1310 1315 1320 Arg Ser Phe Phe Gly Ser Val Ile Phe Leu Cys Arg Arg Gln Ser 1325 1330 1335 Pro Ala Lys Ala Pro Ile Leu Leu Pro Val Asp Asp Thr His Tyr 1340 1345 1350 Lys Trp Val Asp Ser Leu Lys Glu Ile Leu Ala Asp Ser Ser Glu 1355 1360 1365 Gln Pro Leu Trp Leu Thr Ala Thr Asn Cys Gly Asn Ser Gly Ile 1370 1375 1380 Leu Gly Met Val Asn Cys Leu Arg Leu Glu Ala Glu Gly His Arg 1385 1390 1395 Ile Arg Cys Val Phe Val Ser Asn Leu Ser Pro Ser Ser Thr Val 1400 1405 1410 Pro Ala Thr Ser Leu Ser Ser Leu Glu Met Gln Lys Ile Ile Glu 1415 1420 1425 Arg Asp Leu Val Met Asn Val Tyr Arg Asp Gly Lys Trp Gly Ser 1430 1435 1440 Phe Arg His Leu Pro Leu Gln Gln Ala Gln Pro Gln Glu Leu Thr 1445 1450 1455 Glu Tyr Ala Tyr Val Asn Val Leu Thr Arg Gly Asp Leu Ser Ser 1460 1465 1470 Leu Arg Trp Ile Val Ser Pro Leu Arg His Phe Gln Thr Thr Asn 1475 1480 1485 Pro Asn Val Gln Leu Cys Lys Val Tyr Tyr Ala Ser Leu Asn Phe 1490 1495 1500 Arg Asp Ile Met Leu Ala Thr Gly Lys Leu Ser Pro Asp Ala Ile 1505 1510 1515 Pro Gly Asn Trp Thr Leu Gln Gln Cys Met Leu Gly Met Glu Phe 1520 1525 1530 Ser Gly Arg Asp Leu Ala Gly Arg Arg Val Met Gly Leu Leu Pro 1535 1540 1545 Ala Lys Gly Leu Ala Thr Val Val Asp Cys Asp Lys Arg Phe Leu 1550 1555 1560 Trp Glu Val Pro Glu Asn Trp Thr Leu Glu Glu Ala Ala Ser Val 1565 1570 1575 Pro Val Val Tyr Ala Thr Ala Tyr Tyr Ala Leu Val Val Arg Gly 1580 1585 1590 Gly Met Lys Lys Gly Glu Ser Val Leu Ile His Ser Gly Ser Gly 1595 1600 1605 Gly Val Gly Ala Ser Ser His Cys His Arg Leu Glu His Gly Leu 1610 1615 1620 Ala Arg Val Phe Ala Thr Val Gly Ser Ala Glu Lys Arg Glu Tyr 1625 1630 1635 Leu Gln Ala Arg Phe Pro Gln Leu Asp Ala Asn Ser Phe Ala Ser 1640 1645 1650 Ser Arg Asn Thr Thr Phe Glu Gln His Ile Leu Arg Val Thr Asn 1655 1660 1665 Gly Lys Gly Val Asn Leu Val Leu Asn Ser Leu Ala Glu Glu Lys 1670 1675 1680 Leu Gln Ala Ser Leu Arg Cys Leu Ala Gln His Gly Arg Phe Leu 1685 1690 1695 Glu Ile Gly Lys Phe Asp Leu Ser Asn Asn Ser Gln Leu Gly Met 1700 1705 1710 Ala Leu Phe Leu Lys Asn Val Ala Phe His Gly Ile Leu Leu Asp 1715 1720 1725 Ser Ile Phe Glu Glu Gly Asn Gln Glu Trp Glu Val Val Ser Glu 1730 1735 1740 Leu Leu Thr Lys Gly Ile Lys Asp Gly Val Val Lys Pro Leu Arg 1745 1750 1755 Thr Thr Val Phe Gly Lys Glu Glu Val Glu Ala Ala Phe Arg Phe 1760 1765 1770 Met Ala Gln Gly Lys His Ile Gly Lys Val Met Ile Lys Ile Gln 1775 1780 1785 Glu Glu Glu Lys Gln Tyr Pro Leu Arg Ser Glu Pro Val Lys Leu 1790 1795 1800 Ser Ala Ile Ser Arg Thr Ser Cys Pro Pro Thr Lys Ser Tyr Ile 1805 1810 1815 Ile Thr Gly Gly Leu Gly Gly Phe Gly Leu Glu Leu Ala Gln Trp 1820 1825 1830 Leu Ile Glu Arg Gly Ala Gln Lys Leu Val Leu Thr Ser Arg Ser 1835 1840 1845 Gly Ile Arg Thr Gly Tyr Gln Ala Lys Cys Val Arg Glu Trp Lys 1850 1855 1860 Ala Leu Gly Ile Gln Val Leu Val Ser Thr Ser Asp Val Gly Thr 1865 1870 1875 Leu Glu Gly Thr Gln Leu Leu Ile Glu Glu Ala Leu Lys Leu Gly 1880 1885 1890 Pro Val Gly Gly Ile Phe Asn Leu Ala Val Val Leu Lys Asp Ala 1895 1900 1905 Met Ile Glu Asn Gln Thr Pro Glu Leu Phe Trp Glu Val Asn Lys 1910 1915 1920 Pro Lys Tyr Ser Gly Thr Leu His Leu Asp Trp Val Thr Arg Lys 1925 1930 1935 Lys Cys Pro Asp Leu Asp Tyr Phe Val Val Phe Ser Ser Val Ser 1940 1945 1950 Cys Gly Arg Gly Asn Ala Gly Gln Ser Asn Tyr Gly Phe Ala Asn 1955 1960 1965 Ser Ala Met Glu Arg Ile Cys Glu Gln Arg His His Asp Gly Leu 1970 1975 1980 Pro Gly Leu Ala Val Gln Trp Gly Ala Ile Gly Asp Val Gly Ile 1985 1990 1995 Leu Lys Ala Met Gly Asn Arg Glu Val Val Ile Gly Gly Thr Val 2000 2005 2010 Leu Gln Gln Ile Ser Ser Cys Leu Glu Val Leu Asp Met Phe Leu 2015 2020 2025 Asn Gln Pro His Pro Val Met Ser Ser Phe Val Leu Ala Glu Lys 2030 2035 2040 Val Ser Val Lys Ser Glu Gly Gly Ser Gln Arg Asp Leu Val Glu 2045 2050 2055 Ala Val Ala His Ile Leu Gly Val Arg Asp Val Ser Ser Leu Asn 2060 2065 2070 Ala Glu Ser Ser Leu Ala Asp Leu Gly Leu Asp Ser Leu Met Gly 2075 2080 2085 Val Glu Val Arg Gln Thr Leu Glu Arg Asp Tyr Asp Ile Val Met 2090 2095 2100 Thr Met Arg Glu Ile Arg Leu Leu Thr Ile Asn Lys Leu Arg Glu 2105 2110 2115 Leu Ser Ser Lys Thr Gly Thr Ala Glu Glu Leu Lys Pro Ser Gln 2120 2125 2130 Val Leu Lys Thr Gly Pro Gly Glu Pro Pro Lys Leu Asp Leu Asn 2135 2140 2145 Asn Leu Leu Val Asn Pro Glu Gly Pro Thr Ile Thr Arg Leu Asn 2150 2155 2160 Glu Val Gln Ser Thr Glu Arg Pro Leu Phe Leu Val His Pro Ile 2165 2170 2175 Glu Gly Ser Ile Ala Val Phe Tyr Thr Leu Ala Ser Lys Leu His 2180 2185 2190 Met Pro Cys Tyr Gly Leu Gln Cys Thr Lys Ala Ala Pro Leu Asp 2195 2200 2205 Ser Ile Gln Ser Leu Ala Ser Tyr Tyr Ile Asp Cys Met Lys Gln 2210 2215 2220 Ile Gln Pro Glu Gly Pro Tyr Arg Ile Ala Gly Tyr Ser Phe Gly 2225 2230 2235 Ala Cys Val Ala Phe Glu Met Cys Ser Gln Leu Gln Ala Gln Gln 2240 2245 2250 Asn Ala Ser His Ala Leu Asn Ser Leu Phe Leu Phe Asp Gly Ser 2255 2260 2265 His Ser Phe Val Ala Ala Tyr Thr Gln Cys Phe Ser Phe Ser Leu 2270 2275 2280 Phe Gln Ser Tyr Arg Ala Lys Leu Thr Gln Gly Asn Glu Ala Ala 2285 2290 2295 Leu Glu Thr Glu Ala Leu Cys Ala Phe Val Gln Gln Phe Thr Gly 2300 2305 2310 Ile Glu Tyr Asn Lys Leu Leu Glu Ile Leu Leu Pro Leu Glu Asp 2315 2320 2325 Leu Glu Ala Arg Val Asn Ala Ala Ala Asp Leu Ile Thr Gln Ile 2330 2335 2340 His Lys Asn Ile Asn Arg Glu Ala Leu Ser Phe Ala Ala Ala Ser 2345 2350 2355 Phe Tyr His Lys Leu Lys Ala Ala Asp Lys Tyr Ile Pro Glu Ser 2360 2365 2370 Lys Tyr His Gly Asn Val Thr Leu Met Arg Ala Lys Thr His Asn 2375 2380 2385 Glu Tyr Glu Glu Gly Leu Gly Gly Asp Tyr Arg Leu Ser Glu Val 2390 2395 2400 Cys Asp Gly Lys Val Ser Val His Ile Ile Glu Gly Asp His Arg 2405 2410 2415 Thr Leu Leu Glu Gly Asp Gly Val Glu Ser Ile Ile Gly Ile Ile 2420 2425 2430 His Gly Ser Leu Ala Glu Pro Arg Val Ser Val Arg Glu Gly 2435 2440 2445 31 2796 PRT Mycobacterium bovis 31 Met Gly Thr Arg Thr Gly Gly Arg Gly Pro Gly Ser Val Arg Gln Ala 1 5 10 15 Pro Asp Val Gly Arg Arg Val Gly Ala Arg Arg Val Ala Tyr Pro Asp 20 25 30 Arg Gly Asp Pro Gly Ala Gly Pro Ser Arg His Gly Pro Arg Gly His 35 40 45 Pro Ala Gly Arg His Gly Gly His Ser Gln Gly Val Leu Ala Val Glu 50 55 60 Ala Leu Lys Ala Gly Gly Ala Arg Asp Val Glu Leu Phe Ala Leu Ala 65 70 75 80 Gln Leu Ile Gly Ala Ala Gly Thr Leu Val Ala Arg Arg Arg Glu Phe 85 90 95 Pro Ser Trp Ala Ile Ala Pro Met Val Ser Val Thr Asn Ala Asp Pro 100 105 110 Glu Arg Ile Gly Arg Leu Leu Asp Glu Phe Ala Gln Asp Val Arg Thr 115 120 125 Val Leu Pro Pro Val Leu Ser Ile Arg Asn Gly Arg Arg Ala Val Val 130 135 140 Ile Thr Gly Thr Pro Glu Gln Leu Ser Arg Phe Glu Leu Tyr Cys Arg 145 150 155 160 Gln Ile Ser Glu Lys Glu Glu Ala Asp Arg Lys Asn Lys Val Arg Gly 165 170 175 Gly Asp Val Phe Ser Pro Val Phe Glu Pro Val Gln Val Glu Val Gly 180 185 190 Phe His Thr Pro Arg Leu Ser Asp Gly Ile Asp Ile Val Ala Gly Trp 195 200 205 Ala Glu Lys Ala Gly Leu Asp Val Ala Leu Ala Arg Glu Leu Ala Asp 210 215 220 Ala Ile Leu Ile Arg Lys Val Asp Trp Val Asp Glu Ile Thr Arg Val 225 230 235 240 His Arg Ala Gly Ala Arg Trp Ile Leu Asp Leu Gly Pro Gly Asp Ile 245 250 255 Leu Thr Arg Leu Thr Ala Pro Val Ile Arg Gly Leu Gly Ile Gly Ile 260 265 270 Val Pro Ala Arg Thr Arg Gly Gly Gln Arg Asn Leu Phe Thr Val Gly 275 280 285 Ala Thr Pro Glu Val Ala Arg Ala Trp Ser Ser Tyr Ala Pro Thr Val 290 295 300 Val Arg Leu Pro Asp Gly Arg Val Lys Leu Ser Thr Lys Phe Thr Arg 305 310 315 320 Leu Thr Arg Arg Ser Pro Ile Leu Leu Ala Gly Met Thr Pro Thr Thr 325 330 335 Val Asp Ala Lys Ile Val Ala Ala Ala Ala Asn Gly Arg His Trp Ala 340 345 350 Glu Leu Ala Ala Arg Gly Arg Ser Pro Lys Arg Ser Ser Val Thr Ala 355 360 365 Ser Asn Lys Trp Pro Ala Cys Ser Ser Arg Ala Ala Pro Ile Ser Ser 370 375 380 Thr Arg Cys Ser Ser Ile Pro Thr Cys Glu Ala Ser Gly Gly Arg Gln 385 390 395 400 Ala Val Gly Ala Glu Gly Pro Pro Val Arg Arg Arg Asp Arg Arg Arg 405 410 415 Gly Asp Gln Arg Arg His Pro Arg Pro Arg Arg Gly Arg Arg Ala Asp 420 425 430 Arg Arg Thr Gly Arg His Arg His Gln Pro Arg Arg Val Gln Thr Arg 435 440 445 Asp His Arg Ala Asp Pro Leu Gly Asp Ser His Arg His Arg Gly Ala 450 455 460 His Gln Ala Gly Asp His Ala Arg Arg Gly Pro Gly Ala Pro Ala Gly 465 470 475 480 Thr Ile Pro Gly Arg Ile Ser His Leu Leu Leu Ala Thr Tyr Ser Ala 485 490 495 Asp Arg Ala Pro Arg Gln His His Val Cys Val Gly Gly Gly His Leu 500 505 510 Gly Thr Pro Lys Lys Gly Cys Gly Tyr Leu Ser Gly Pro Gly Arg Ser 515 520 525 Val Arg Leu Pro Ile Asp Ala Asp Arg Arg Ile Leu Val Gly Thr Ala 530 535 540 Ala Met Ala Thr Lys Glu Ser Thr Thr Ser Pro Ser Val Lys Arg Met 545 550 555 560 Leu Val Asp Thr Gln Gly Thr Asp Gln Trp Ile Ser Ala Gly Lys Ala 565 570 575 Gln Gly Arg Met Pro Pro Ala Glu Ser Ala Arg Cys Arg His Pro Arg 580 585 590 Asp Arg His Ser Ala Ser Val Arg Arg Cys Ser Thr Arg Trp Pro Val 595 600 605 Thr Arg Arg Arg Ser Arg Ser Val Ala Trp Pro Arg Pro Pro Ser Pro 610 615 620 Thr Cys Arg Arg Arg Arg His Asp Leu Pro Ala Val Ala Ala Gly Ala 625 630 635 640 Thr Ser Asn Trp Pro Ser Gly Lys Ala Thr Arg Pro Pro Thr Pro Pro 645 650 655 Arg Trp Ala Ala Arg Gly Trp Pro Thr Leu Ala Gly Pro Leu Arg Ala 660 665 670 Asp Ala Ala Ala Cys Arg Ser Pro Val Ala Pro Thr Gly Phe Arg Pro 675 680 685 Asp Pro Asp Ala Ile His Arg Cys Trp Pro Ala Gly Gln Ser Ala Ala 690 695 700 Ala Ile Ala Ala Leu Val Ala Arg Tyr Pro Asp Ala Glu Thr Val Gln 705 710 715 720 Leu His Pro Ala Asp Val Pro Phe Phe Val Thr Leu Cys Lys Thr Leu 725 730 735 Gly Lys Pro Val Asn Phe Val Pro Ala Ile Asp Leu Val Val Arg Ala 740 745 750 Gly Gly Ala Ala Thr Arg Cys Gly Arg Pro Thr Thr Pro Ala Thr Thr 755 760 765 Pro Met Arg Cys Ala Ser Phe Arg Ala Arg Val Gly Ser Arg Ile Thr 770 775 780 Arg Met Asp Glu Pro Val Gly Glu Leu Leu Asp Ala Phe Glu Gln Ala 785 790 795 800 Ala Ile Asp Glu Val Leu Gly Ala Gly Val Glu Pro Lys Asp Val Ala 805 810 815 Ser Gly Arg Leu Gly Arg Ala Asp Val Ala Gly Pro Leu Ala Val Val 820 825 830 Leu Asp Ala Pro Asp Val Arg Trp Ala Gly Arg Thr Val Thr Asn Pro 835 840 845 Val His Arg Ile Arg Asp Pro Ala Glu Trp Gln Val His Asp Gly Pro 850 855 860 Glu Asn Pro Arg Ala Ala His Ser Ser Thr Gly Ala Arg Leu Gln Thr 865 870 875 880 His Gly Asp Asp Val Ala Leu Ser Val Ala Arg Leu Gly His Leu Gly 885 890 895 Arg His Pro Ile His Val Ala Gly Gln His Arg Arg Trp Arg His Pro 900 905 910 Gly Asp Arg His Arg Gly Arg His His Ala Met Arg Thr Val Leu Arg 915 920 925 Ser Pro Pro Val Ser Thr Ala Arg Ser Ser Cys Cys Gly Gly Gln Arg 930 935 940 Asp Gly His Phe Asp Gly Gly Leu Ala Pro Arg Ala Cys Cys Arg Pro 945 950 955 960 His Arg His Arg His Val Arg Cys Ala Leu Ala Pro Ser Leu Thr Asn 965 970 975 Val Pro Thr Arg Leu Val Gly Pro Cys Trp Pro Ala Val Phe Ala Ala 980 985 990 Ile Gly Ser Ala Val Thr Asp Thr Gly Glu Pro Val Val Glu Gly Leu 995 1000 1005 Leu Ser Leu Val His Leu Asp Thr Arg Pro Arg Val Val Gly Gln 1010 1015 1020 Leu Pro Thr Val Pro Ala Gln Leu Thr Val Thr Gln Arg Leu Pro 1025 1030 1035 Thr Gln Pro Ile Arg Thr Trp Ala Ala Ser Cys Arg Ser Arg Ser 1040 1045 1050 Ser Phe Thr Ala Trp Arg Arg Asp Arg His Ser Arg Gly Ala Ile 1055 1060 1065 Arg Asp Pro Gly Ser His Arg Phe Ala Glu Leu Asp Arg Arg Glu 1070 1075 1080 Pro Val Ala Arg Cys Arg Glu Arg His Arg His Pro Ala Arg Arg 1085 1090 1095 Arg Arg Asp Val Thr Ile Thr Ala Pro Val Asp Met Arg Pro Phe 1100 1105 1110 Ala Val Val Ser Gly Asp His Asn Pro Ile His Thr Asp Arg Ala 1115 1120 1125 Ala Ala Ala Cys Arg Pro Gly Val Ala Asp Arg Ala Arg His Val 1130 1135 1140 Ala Val Gly Arg Gly Ala Thr Arg Gly Asp Arg His Arg Arg Ala 1145 1150 1155 Gly Pro Pro Pro Ala Arg Leu Val Gly Trp Thr Ala Arg Phe Leu 1160 1165 1170 Gly Met Val Ala Pro Ala Thr Arg Trp Thr Ser Gly Arg Ala Arg 1175 1180 1185 Arg Ile Asp Gln Gly Ala Glu Ile Val Asp Val Ala Ala Arg Val 1190 1195 1200 Gly Ser Asp Leu Val Met Ser Ala Ser Ala Arg Leu Ala Ala Pro 1205 1210 1215 Lys Thr Val Tyr Ala Phe Pro Gly Gln Gly Ile Gln His Lys Gly 1220 1225 1230 Met Gly Met Glu Val Arg Ala Ala Pro Arg Arg Pro Ala Arg Cys 1235 1240 1245 Gly Thr Pro Arg Thr Ser Ser Pro Ala Thr Pro Trp Ala Ser Arg 1250 1255 1260 Tyr Cys Thr Trp Ser Ala Thr Thr Arg Pro Ala Ser Ser Pro Ala 1265 1270 1275 Val Cys Thr Thr Thr Thr Asp Gly Val Leu Tyr Leu Thr Gln Phe 1280 1285 1290 Thr Gln Val Ala Met Ala Thr Val Ala Ala Gly Gln Val Ala Glu 1295 1300 1305 Met Arg Glu Gln Gly Ala Phe Val Glu Gly Ala Ile Ala Cys Gly 1310 1315 1320 His Ser Val Gly Glu Tyr Thr Ala Leu Ala Cys Val Thr Gly Ile 1325 1330 1335 Tyr Gln Leu Glu Ala Leu Leu Glu Met Val Phe His Arg Gly Ser 1340 1345 1350 Lys Met His Asp Ile Val Pro Arg Asp Glu Leu Gly Arg Ser Asn 1355 1360 1365 Tyr Arg Leu Ser Ala Ile Arg Pro Ser Gln Ile Asp Leu Asp Asp 1370 1375 1380 Ala Asp Val Pro Ala Phe Val Ala Gly Ile Ala Glu Ser Thr Gly 1385 1390 1395 Glu Phe Leu Glu Ile Glu Asn Phe Asn Leu Gly Gly Ser Gln Tyr 1400 1405 1410 Ala Ile Ala Gly Thr Val Arg Gly Leu Glu Ala Leu Glu Ala Glu 1415 1420 1425 Val Glu Arg Arg Arg Glu Leu Thr Gly Gly Arg Arg Ser Phe Ile 1430 1435 1440 Leu Val Pro Gly Ile Asp Val Pro Phe His Ser Arg Val Leu Arg 1445 1450 1455 Val Gly Val Ala Glu Phe Arg Arg Ser Leu Asp Arg Val Met Arg 1460 1465 1470 Pro Thr Arg Thr Arg Pro Asp His Arg Ala Leu His Ser Gln Pro 1475 1480 1485 Gly Ala Ala Glu Val Gln Pro Trp Thr Ala Thr Ser Ser Arg Lys 1490 1495 1500 Ser Gly Ile Trp Cys Pro Ala Glu Pro Leu Asp Glu Ile Leu Ala 1505 1510 1515 Asp Tyr Asp Thr Trp Leu Arg Asp Asp Arg Arg Asp Gly Ala His 1520 1525 1530 Gly Val His Arg Ala Ala Gly Met Ala Ile Arg Gln Pro Gly Ala 1535 1540 1545 Leu Asp Arg Asp Ala Gly Ser Ala Val His Arg Gly Gly Ala Gly 1550 1555 1560 Gly Leu Gly Val Glu Arg Phe Val Glu Ile Gly Val Lys Ser Ser 1565 1570 1575 Pro Thr Val Ala Gly Ser Cys His Gln His Pro Gln Thr Ala Arg 1580 1585 1590 Ile Arg Pro Gln His Ser Glu Val Leu Asn Ala Glu Arg Asp Ala 1595 1600 1605 Arg Cys Cys Ser Pro Pro Thr Pro Thr Arg Ser Arg Ser Arg Arg 1610 1615 1620 Lys Thr Ser Arg Ser Arg Asn Arg Pro Arg Arg Thr Ser Ser Arg 1625 1630 1635 Lys Pro Pro Pro Ser Arg Arg Pro Leu Arg Arg Arg Ala Arg Val 1640 1645 1650 Pro Thr Ile Trp Phe Ser Thr Pro Pro Met Pro Arg Cys Val Ile 1655 1660 1665 Ala Leu Ser Ala Lys Met Arg Ile Asp Gln Ile Glu Glu Leu Asp 1670 1675 1680 Ser Ile Glu Ser Ile Thr Asp Gly Ala Ser Ser Arg Arg Asn Gln 1685 1690 1695 Leu Leu Val Asp Leu Gly Ser Glu Leu Asn Leu Gly Ala Ile Glu 1700 1705 1710 Arg Arg Arg Arg Ile Gly Pro Gly Arg Ser Ala Leu Thr Gly Asp 1715 1720 1725 Gln Thr Gly Ala His Leu Gln Arg Tyr Gly Pro Val Leu Ser Asp 1730 1735 1740 Ala Ile Asn Asp His Val Arg Thr Val Leu Gly Pro Ser Gly Lys 1745 1750 1755 Arg Pro Gly Ala Ile Ala Glu Arg Val Lys Lys Thr Trp Glu Leu 1760 1765 1770 Gly Glu Ala Gly Pro Ser Met Ser Pro Ser Arg Ser Arg Trp Ala 1775 1780 1785 Pro Ala Arg Ala Ala Ala Phe Ala Ala Ala Pro Trp Ala Thr Cys 1790 1795 1800 Thr Arg Ala Arg Trp Pro Met Pro Pro Pro Ser Thr Arg Ser Ser 1805 1810 1815 Thr Arg Arg Ser His Arg Trp Pro Arg Pro Gly Arg Phe Gly Ser 1820 1825 1830 Ala Ala Ser Ala Gly Ser Gly Gly Ala Thr Ile Asp Ala Ala Ala 1835 1840 1845 Leu Ser Glu Phe Thr Asp Gln Ile Thr Gly Arg Glu Gly Val Leu 1850 1855 1860 Pro Pro Arg Pro Ala Trp Cys Trp Gly Ser Trp Asp Trp Thr Thr 1865 1870 1875 Pro Ser Thr Val Ala Gly Arg Pro Asp Ser Glu Leu Ile Asp Leu 1880 1885 1890 Val Thr Ala Glu Leu Gly Arg Thr Gly Arg Gly Trp Trp His Arg 1895 1900 1905 Cys Ser Thr Pro Arg Arg Pro Ser Tyr Ser Thr Thr Ala Gly Gln 1910 1915 1920 Arg Pro Arg Gly Pro Gly Glu Ala Val Ala Asp Arg Arg Lys Asp 1925 1930 1935 Arg Arg Arg His Arg Arg Arg Leu Ala Ala Leu Ala Glu Arg Phe 1940 1945 1950 Glu Gly Ala Ala Thr Ser Trp Arg Pro Arg Leu Pro Gly Gly Lys 1955 1960 1965 Val Ser Arg Ser Arg Gly Pro Ala Asp Pro Cys Ile Ala Val Arg 1970 1975 1980 Pro His Ala Ala Gly Ala Glu Asn Pro Glu Pro Arg Val Arg Arg 1985 1990 1995 Arg Ser Cys Arg Gly Asp Arg Arg Phe Glu Gly Phe Asp Arg Arg 2000 2005 2010 Val Gly Gly Gly Ser Ala Ala Arg Arg Gly Ala Thr Val Ile Ala 2015 2020 2025 Thr Thr Ser Lys Leu Asp Glu Glu Arg Leu Arg Phe Tyr Arg Thr 2030 2035 2040 Leu Tyr Arg Asp His Ala Arg Tyr Gly Ala Ala Leu Trp Leu Val 2045 2050 2055 Ala Ala Asn Met Ala Ser Tyr Ser Asp Val Asp Ala Leu Val Glu 2060 2065 2070 Trp Ile Gly Thr Glu Gln Thr Glu Ser Leu Gly Pro Gln Ser Ile 2075 2080 2085 His Ile Lys Asp Ala Gln Thr Pro Thr Leu Leu Phe Arg Ser Arg 2090 2095 2100 Arg Thr Arg Val Gly Thr Val Gly Gly Arg Phe Ala Arg Arg Asp 2105 2110 2115 Gly Asp Glu Ser Ala Ala Val Ala Val Gln Arg Leu Ile Gly Gly 2120 2125 2130 Leu Ser Thr Ile Gly Ala Glu Arg Asp Met Pro Ser Arg Leu Glu 2135 2140 2145 Arg Gly Ala Ala Arg Leu Ala Gln Pro Trp His Val Arg Arg Arg 2150 2155 2160 Arg Ala Leu Arg Arg Ser Gln Val Arg Ala Gly Cys Arg Gly Asp 2165 2170 2175 Ala Leu Ala Arg Arg Val Val Leu Gly Gly Thr Gly Gln Pro Gly 2180 2185 2190 Ala Arg Ala His Arg Leu Asp Pro Arg His Arg Ala Asp Gly Pro 2195 2200 2205 Gln Arg Cys His Arg Gly Arg Arg Arg Arg Gly Arg Gly His His 2210 2215 2220 Leu Leu Asp Arg Arg Asp Gly Ala Ala Ala Ala Arg Pro Val Ser 2225 2230 2235 Cys Gly Ile Gln Gly Gly Cys Gly Arg Ser Pro Ile Lys Ala Asp 2240 2245 2250 Leu Thr Gly Gly Leu Pro Arg Pro Thr Ser Thr Trp Pro Ser Trp 2255 2260 2265 Arg Pro Arg Arg Ala Ser Arg Cys Arg Gln Arg Arg Pro Ser Thr 2270 2275 2280 Arg Thr Pro Arg Pro Leu Ala Pro Ser Pro Arg Cys Arg Arg Arg 2285 2290 2295 Pro Gly Phe Thr Pro Ala Pro Pro Pro Gln Trp Asp Asp Leu Asp 2300 2305 2310 Val Asp Pro Ala Asp Leu Val Val Ile Val Gly Gly Arg Glu Ile 2315 2320 2325 Gly Pro Tyr Gly Ser Ser Arg Thr Arg Phe Glu Met Glu Val Glu 2330 2335 2340 Asn Glu Leu Ser Ala Ala Gly Val Leu Glu Leu Ala Trp Thr Thr 2345 2350 2355 Gly Leu Ile Ala Gly Arg Arg Pro Ala Thr Arg Leu Val Arg His 2360 2365 2370 Arg Ile Arg Arg Asn Gly Arg Arg Ile Arg Val Gly Ala Ala Leu 2375 2380 2385 His Asp Ala Val Val Gln Arg Val Gly Ile Arg Glu Phe Val Asp 2390 2395 2400 Asp Gly Ala Ile Asp Pro Asp His Ala Ser Pro Leu Leu Val Ser 2405 2410 2415 Val Phe Leu Glu Lys Asp Phe Ala Phe Val Val Ser Ser Glu Ala 2420 2425 2430 Asp Ala Arg Ala Phe Val Glu Phe Asp Pro Glu His Thr Val Ile 2435 2440 2445 Arg Pro Val Pro Asp Ser Thr Asp Trp Gln Val Ile Arg Lys Ala 2450 2455 2460 Gly Thr Glu Ile Arg Val Pro Arg Lys Thr Lys Leu Ser Arg Val 2465 2470 2475 Val Gly Gly Gln Ile Pro Thr Gly Phe Asp Pro Thr Val Trp Gly 2480 2485 2490 Ile Ser Ala Asp Met Ala Gly Ser Ile Asp Arg Leu Ala Val Trp 2495 2500 2505 Asn Met Trp Arg Thr Val Asp Arg Phe Leu Ser Ser Gly Phe Ser 2510 2515 2520 Pro Ala Glu Val Met Arg Tyr Val His Pro Ser Leu Val Ala Asn 2525 2530 2535 Thr Gln Gly Thr Gly Met Gly Gly Gly Thr Ser Met Gln Thr Met 2540 2545 2550 Tyr His Gly Asn Leu Leu Gly Arg Asn Lys Pro Asn Asp Ile Phe 2555 2560 2565 Gln Glu Val Leu Pro Ile Ser Phe Ala Ala His Val Val Gln Ser 2570 2575 2580 Tyr Val Gly Ser Tyr Gly Ala Met Ile His Pro Val Ala Ala Cys 2585 2590 2595 Ala Thr Ala Ala Val Ser Val Glu Glu Gly Val Asp Lys Ile Arg 2600 2605 2610 Leu Gly Arg Leu Asn Trp Trp Ser Ala Ala Val Asp Asp Leu Thr 2615 2620 2625 Leu Glu Gly Ile Ile Gly Phe Gly Asp Met Ala Ala Thr Ala Asp 2630 2635 2640 Thr Ser Met Met Arg Gly Arg Gly Ile His Asp Ser Lys Phe Ser 2645 2650 2655 Arg Pro Asn Asp Arg Arg Arg Leu Ala Ser Ser Lys Pro Lys Ala 2660 2665 2670 Ala Gly Arg Ser Cys Trp Ala Arg Gly Pro Gly Ala Ala Asp Gly 2675 2680 2685 Ala Ala Gly Ala Gly Gly Gly Gly Phe Ala Gln Ser Phe Gly Asp 2690 2695 2700 Gly Val His Thr Ser Ile Arg Pro Gly Pro Gly Arg Ala Gly Gly 2705 2710 2715 Gly Ala Arg Arg Gln Gly Phe Ser Cys Gly Gly Arg Trp Pro Ser 2720 2725 2730 Cys Val Ala Ala Asp Asp Val Ala Val Ile Ser Lys His Asp Thr 2735 2740 2745 Ser Thr Leu Ala Asn Asp Pro Asn Glu Thr Glu Leu His Glu Arg 2750 2755 2760 Leu Ala Asp Ala Leu Gly Arg Ser Glu Gly Ala Pro Leu Phe Val 2765 2770 2775 Val Ser Gln Lys Ser Leu Thr Gly Gln Pro Arg Ala Ala Arg Arg 2780 2785 2790 Ser Ser Arg 2795 32 675 DNA Bacillus subtilis 32 atgaagattt acggaattta tatggaccgc ccgctttcac aggaagaaaa tgaacggttc 60 atgactttca tatcacctga aaaacgggag aaatgccgga gattttatca taaagaagat 120 gctcaccgca ccctgctggg agatgtgctc gttcgctcag tcataagcag gcagtatcag 180 ttggacaaat ccgatatccg ctttagcacg caggaatacg ggaagccgtg catccctgat 240 cttcccgacg ctcatttcaa catttctcac tccggccgct gggtcattgg tgcgtttgat 300 tcacagccga tcggcataga tatcgaaaaa acgaaaccga tcagccttga gatcgccaag 360 cgcttctttt caaaaacaga gtacagcgac cttttagcaa aagacaagga cgagcagaca 420 gactattttt atcatctatg gtcaatgaaa gaaagcttta tcaaacagga aggcaaaggc 480 ttatcgcttc cgcttgattc cttttcagtg cgcctgcatc aggacggaca agtatccatt 540 gagcttccgg acagccattc cccatgctat atcaaaacgt atgaggtcga tcccggctac 600 aaaatggctg tatgcgccgc acaccctgat ttccccgagg atatcacaat ggtctcgtac 660 gaagagcttt tataa 675 33 224 PRT Bacillus subtilis 33 Met Lys Ile Tyr Gly Ile Tyr Met Asp Arg Pro Leu Ser Gln Glu Glu 1 5 10 15 Asn Glu Arg Phe Met Thr Phe Ile Ser Pro Glu Lys Arg Glu Lys Cys 20 25 30 Arg Arg Phe Tyr His Lys Glu Asp Ala His Arg Thr Leu Leu Gly Asp 35 40 45 Val Leu Val Arg Ser Val Ile Ser Arg Gln Tyr Gln Leu Asp Lys Ser 50 55 60 Asp Ile Arg Phe Ser Thr Gln Glu Tyr Gly Lys Pro Cys Ile Pro Asp 65 70 75 80 Leu Pro Asp Ala His Phe Asn Ile Ser His Ser Gly Arg Trp Val Ile 85 90 95 Gly Ala Phe Asp Ser Gln Pro Ile Gly Ile Asp Ile Glu Lys Thr Lys 100 105 110 Pro Ile Ser Leu Glu Ile Ala Lys Arg Phe Phe Ser Lys Thr Glu Tyr 115 120 125 Ser Asp Leu Leu Ala Lys Asp Lys Asp Glu Gln Thr Asp Tyr Phe Tyr 130 135 140 His Leu Trp Ser Met Lys Glu Ser Phe Ile Lys Gln Glu Gly Lys Gly 145 150 155 160 Leu Ser Leu Pro Leu Asp Ser Phe Ser Val Arg Leu His Gln Asp Gly 165 170 175 Gln Val Ser Ile Glu Leu Pro Asp Ser His Ser Pro Cys Tyr Ile Lys 180 185 190 Thr Tyr Glu Val Asp Pro Gly Tyr Lys Met Ala Val Cys Ala Ala His 195 200 205 Pro Asp Phe Pro Glu Asp Ile Thr Met Val Ser Tyr Glu Glu Leu Leu 210 215 220 34 714 DNA Brevibacillus brevis 34 atgatagaaa tgttatttgt aaaggttcca aacgaaatcg ataggcatgt gtttaacttc 60 ttgtcatcaa atgtgagtaa ggaaaaacag caggcgtttg ttcgatacgt taatgtgaaa 120 gatgcttatc gttctctttt aggggaattg cttattagaa aatatttgat acaagtatta 180 aacattccta atgaaaacat tctatttagg aaaaatgaat atggaaaacc ttttgttgat 240 ttcgatattc attttaatat ttcccactct gatgaatggg ttgtatgtgc aatttcaaat 300 catcctgttg gaattgatat cgagcgtatt tcggagatag acattaaaat agcagaacaa 360 ttttttcatg aaaatgaata tatatggttg cagtctaaag cccaaaatag tcaagtttct 420 tctttttttg agctttggac tattaaagaa agttatataa aagctattgg taaaggtatg 480 tacataccga ttaattcatt ttggattgat aagaatcaaa cacaaactgt aatttacaaa 540 cagaataaaa aagaacctgt tactatttat gaaccagagt tgtttgaggg ctacaagtgt 600 tcttgttgtt ctttgttttc ttctgtaacg aacttgtcta ttactaaatt gcaagtgcaa 660 gagttatgta atttgtttct agattctaca ttttctgaaa ataataactt ttag 714 35 237 PRT Brevibacillus brevis 35 Met Ile Glu Met Leu Phe Val Lys Val Pro Asn Glu Ile Asp Arg His 1 5 10 15 Val Phe Asn Phe Leu Ser Ser Asn Val Ser Lys Glu Lys Gln Gln Ala 20 25 30 Phe Val Arg Tyr Val Asn Val Lys Asp Ala Tyr Arg Ser Leu Leu Gly 35 40 45 Glu Leu Leu Ile Arg Lys Tyr Leu Ile Gln Val Leu Asn Ile Pro Asn 50 55 60 Glu Asn Ile Leu Phe Arg Lys Asn Glu Tyr Gly Lys Pro Phe Val Asp 65 70 75 80 Phe Asp Ile His Phe Asn Ile Ser His Ser Asp Glu Trp Val Val Cys 85 90 95 Ala Ile Ser Asn His Pro Val Gly Ile Asp Ile Glu Arg Ile Ser Glu 100 105 110 Ile Asp Ile Lys Ile Ala Glu Gln Phe Phe His Glu Asn Glu Tyr Ile 115 120 125 Trp Leu Gln Ser Lys Ala Gln Asn Ser Gln Val Ser Ser Phe Phe Glu 130 135 140 Leu Trp Thr Ile Lys Glu Ser Tyr Ile Lys Ala Ile Gly Lys Gly Met 145 150 155 160 Tyr Ile Pro Ile Asn Ser Phe Trp Ile Asp Lys Asn Gln Thr Gln Thr 165 170 175 Val Ile Tyr Lys Gln Asn Lys Lys Glu Pro Val Thr Ile Tyr Glu Pro 180 185 190 Glu Leu Phe Glu Gly Tyr Lys Cys Ser Cys Cys Ser Leu Phe Ser Ser 195 200 205 Val Thr Asn Leu Ser Ile Thr Lys Leu Gln Val Gln Glu Leu Cys Asn 210 215 220 Leu Phe Leu Asp Ser Thr Phe Ser Glu Asn Asn Asn Phe 225 230 235 36 648 DNA Escherichia coli 36 ttgtcatcag tctcgaatat ggtcgatatg aaaactacgc atacctccct cccctttgcc 60 ggacatacgc tgcattttgt tgagttcgat ccggcgaatt tttgtgagca ggatttactc 120 tggctgccgc actacgcaca actgcaacac gctggacgta aacgtaaaac agagcattta 180 gccggacgga tcgctgctgt ttatgctttg cgggaatatg gctataaatg tgtgcccgca 240 atcggcgagc tacgccaacc tgtctggcct gcggaggtat acggcagtat tagccactgt 300 gggactacgg cattagccgt ggtatctcgt caaccgattg gcattgatat agaagaaatt 360 ttttctgtac aaaccgcaag agaattgaca gacaacatta ttacaccagc ggaacacgag 420 cgactcgcag actgcggttt agccttttct ctggcgctga cactggcatt ttccgccaaa 480 gagagcgcat ttaaggcaag tgagatccaa actgatgcag gttttctgga ctatcagata 540 attagctgga ataaacagca ggtcatcatt catcgtgaga atgagatgtt tgctgtgcac 600 tggcagataa aagaaaagat agtcataacg ctgtgccaac acgattaa 648 37 215 PRT Escherichia coli 37 Met Ser Ser Val Ser Asn Met Val Asp Met Lys Thr Thr His Thr Ser 1 5 10 15 Leu Pro Phe Ala Gly His Thr Leu His Phe Val Glu Phe Asp Pro Ala 20 25 30 Asn Phe Cys Glu Gln Asp Leu Leu Trp Leu Pro His Tyr Ala Gln Leu 35 40 45 Gln His Ala Gly Arg Lys Arg Lys Thr Glu His Leu Ala Gly Arg Ile 50 55 60 Ala Ala Val Tyr Ala Leu Arg Glu Tyr Gly Tyr Lys Cys Val Pro Ala 65 70 75 80 Ile Gly Glu Leu Arg Gln Pro Val Trp Pro Ala Glu Val Tyr Gly Ser 85 90 95 Ile Ser His Cys Gly Thr Thr Ala Leu Ala Val Val Ser Arg Gln Pro 100 105 110 Ile Gly Ile Asp Ile Glu Glu Ile Phe Ser Val Gln Thr Ala Arg Glu 115 120 125 Leu Thr Asp Asn Ile Ile Thr Pro Ala Glu His Glu Arg Leu Ala Asp 130 135 140 Cys Gly Leu Ala Phe Ser Leu Ala Leu Thr Leu Ala Phe Ser Ala Lys 145 150 155 160 Glu Ser Ala Phe Lys Ala Ser Glu Ile Gln Thr Asp Ala Gly Phe Leu 165 170 175 Asp Tyr Gln Ile Ile Ser Trp Asn Lys Gln Gln Val Ile Ile His Arg 180 185 190 Glu Asn Glu Met Phe Ala Val His Trp Gln Ile Lys Glu Lys Ile Val 195 200 205 Ile Thr Leu Cys Gln His Asp 210 215 38 741 DNA Streptomyces verticillus 38 gtgatcgccg ccctcctgcc ctcctgggcc gtcaccgaac acgccttcac cgacgccccg 60 gacgacccgg tgagcctcct cttccccgag gaggccgccc acgtcgcccg cgccgtcccc 120 aagcgcctgc acgagttcgc caccgtccgg gtgtgcgccc gcgccgccct cggccggctg 180 ggcctcccgc ccggtccgct gctgcccggc cgacggggcg cgccgagctg gccggacggg 240 gtggtgggga gcatgacgca ctgtcagggc ttccggggcg ccgcggtcgc ccgggccgcc 300 gacgccgcgt cgctcgggat agacgccgag ccgaacgggc cgctcccgga cggcgtcctc 360 gccatggtct cgctgccgtc cgagcgcgag tggctcgccg gactggcggc ccgccggccg 420 gacgtgcact gggaccggct gctgttcagc gccaaggaga gcgtcttcaa ggcgtggtac 480 ccgctgaccg gcctggagct ggacttcgac gaggccgagc tggccgtcga tccggacgcc 540 gggacgttca cggcccggct gctggtgccg ggaccggtgg tcggcggccg tcggctggac 600 gggttcgagg ggcgctgggc ggcgggcgag ggcctcgtcg tcacggccat cgccgtcgcg 660 gcgccggccg gtaccgcgga ggaatcggcg gaaggggccg ggaaggaagc gactgcggac 720 gaccggaccg ccgtcccgta a 741 39 246 PRT Streptomyces verticillus 39 Met Ile Ala Ala Leu Leu Pro Ser Trp Ala Val Thr Glu His Ala Phe 1 5 10 15 Thr Asp Ala Pro Asp Asp Pro Val Ser Leu Leu Phe Pro Glu Glu Ala 20 25 30 Ala His Val Ala Arg Ala Val Pro Lys Arg Leu His Glu Phe Ala Thr 35 40 45 Val Arg Val Cys Ala Arg Ala Ala Leu Gly Arg Leu Gly Leu Pro Pro 50 55 60 Gly Pro Leu Leu Pro Gly Arg Arg Gly Ala Pro Ser Trp Pro Asp Gly 65 70 75 80 Val Val Gly Ser Met Thr His Cys Gln Gly Phe Arg Gly Ala Ala Val 85 90 95 Ala Arg Ala Ala Asp Ala Ala Ser Leu Gly Ile Asp Ala Glu Pro Asn 100 105 110 Gly Pro Leu Pro Asp Gly Val Leu Ala Met Val Ser Leu Pro Ser Glu 115 120 125 Arg Glu Trp Leu Ala Gly Leu Ala Ala Arg Arg Pro Asp Val His Trp 130 135 140 Asp Arg Leu Leu Phe Ser Ala Lys Glu Ser Val Phe Lys Ala Trp Tyr 145 150 155 160 Pro Leu Thr Gly Leu Glu Leu Asp Phe Asp Glu Ala Glu Leu Ala Val 165 170 175 Asp Pro Asp Ala Gly Thr Phe Thr Ala Arg Leu Leu Val Pro Gly Pro 180 185 190 Val Val Gly Gly Arg Arg Leu Asp Gly Phe Glu Gly Arg Trp Ala Ala 195 200 205 Gly Glu Gly Leu Val Val Thr Ala Ile Ala Val Ala Ala Pro Ala Gly 210 215 220 Thr Ala Glu Glu Ser Ala Glu Gly Ala Gly Lys Glu Ala Thr Ala Asp 225 230 235 240 Asp Arg Thr Ala Val Pro 245 40 819 DNA Saccharomyces cerevisiae 40 atggttaaaa cgactgaagt agtaagcgaa gtttcaaagg tggcaggtgt aagaccatgg 60 gcaggtatat tcgttgttga aattcaagag gatatactcg cggatgagtt tacgttcgag 120 gcattaatga gaactttgcc attggcgtct caagccagaa tcctcaataa aaaatcgttt 180 cacgatagat gttcaaatct atgcagccag ctgctgcagt tgtttggctg ctctatagta 240 acgggcttaa attttcaaga gctgaaattt gacaagggca gcttcggtaa gccattctta 300 gacaacaatc gttttcttcc atttagcatg accatcggtg aacaatatgt agctatgttc 360 ctcgtaaaat gtgtaagtac agatgaatac caggatgtcg gaattgatat cgcttctccg 420 tgcaattatg gcgggaggga agagttggag ctatttaaag aagtttttag tgaaagagaa 480 tttaacggtt tactgaaagc gtctgatcca tgcacaatat ttacttactt atggtccttg 540 aaggagtcgt atacaaaatt tactggaact ggccttaaca cagacttgtc actaatagat 600 tttggcgcta tcagcttttt tccggctgag ggagcttcta tgtgcataac tctggatgaa 660 gttccattga ttttccattc tcaatggttc aataacgaaa ttgtcactat ctgtatgcca 720 aagtccatca gtgataaaat caacacgaac agaccaaaat tatataatat cagcttatct 780 acgttgattg attatttcat cgaaaatgat ggtttataa 819 41 272 PRT Saccharomyces cerevisiae 41 Met Val Lys Thr Thr Glu Val Val Ser Glu Val Ser Lys Val Ala Gly 1 5 10 15 Val Arg Pro Trp Ala Gly Ile Phe Val Val Glu Ile Gln Glu Asp Ile 20 25 30 Leu Ala Asp Glu Phe Thr Phe Glu Ala Leu Met Arg Thr Leu Pro Leu 35 40 45 Ala Ser Gln Ala Arg Ile Leu Asn Lys Lys Ser Phe His Asp Arg Cys 50 55 60 Ser Asn Leu Cys Ser Gln Leu Leu Gln Leu Phe Gly Cys Ser Ile Val 65 70 75 80 Thr Gly Leu Asn Phe Gln Glu Leu Lys Phe Asp Lys Gly Ser Phe Gly 85 90 95 Lys Pro Phe Leu Asp Asn Asn Arg Phe Leu Pro Phe Ser Met Thr Ile 100 105 110 Gly Glu Gln Tyr Val Ala Met Phe Leu Val Lys Cys Val Ser Thr Asp 115 120 125 Glu Tyr Gln Asp Val Gly Ile Asp Ile Ala Ser Pro Cys Asn Tyr Gly 130 135 140 Gly Arg Glu Glu Leu Glu Leu Phe Lys Glu Val Phe Ser Glu Arg Glu 145 150 155 160 Phe Asn Gly Leu Leu Lys Ala Ser Asp Pro Cys Thr Ile Phe Thr Tyr 165 170 175 Leu Trp Ser Leu Lys Glu Ser Tyr Thr Lys Phe Thr Gly Thr Gly Leu 180 185 190 Asn Thr Asp Leu Ser Leu Ile Asp Phe Gly Ala Ile Ser Phe Phe Pro 195 200 205 Ala Glu Gly Ala Ser Met Cys Ile Thr Leu Asp Glu Val Pro Leu Ile 210 215 220 Phe His Ser Gln Trp Phe Asn Asn Glu Ile Val Thr Ile Cys Met Pro 225 230 235 240 Lys Ser Ile Ser Asp Lys Ile Asn Thr Asn Arg Pro Lys Leu Tyr Asn 245 250 255 Ile Ser Leu Ser Thr Leu Ile Asp Tyr Phe Ile Glu Asn Asp Gly Leu 260 265 270 42 588 DNA Escherichia coli 42 atggtggacc aggcgcagga caccctgcgc ccgaataaca gattgtcaga tatgcaggca 60 acaatggaac aaacccaggc ctttgaaaac cgtgtgcttg agcgtctgaa tgctggcaaa 120 accgtgcgaa gctttctgat caccgccgtc gagctcctga ccgaggcggt aaatcttctg 180 gtgcttcagg tattccgcaa agacgattac gcggtgaagt atgctgtaga accgttactc 240 gacggcgatg gtccgctggg cgatctttct gtgcgtttaa aactcattta cgggttgggc 300 gtcattaacc gccaggaata cgaagatgcg gaactgctga tggcattgcg tgaagagcta 360 aatcacgacg gcaacgagta cgcctttacc gacgacgaaa tccttggacc ctttggtgaa 420 ctgcactgcg tggcggcgtt accaccgccg ccacagtttg aaccagcaga ctccagtttg 480 tatgcaatgc aaattcagcg ctatcaacag gctgtgcgat caacaatggt cctttcactg 540 actgagctga tttccaaaat cagcttaaaa aaagcctttc aaaagtaa 588 43 195 PRT Escherichia coli 43 Met Val Asp Gln Ala Gln Asp Thr Leu Arg Pro Asn Asn Arg Leu Ser 1 5 10 15 Asp Met Gln Ala Thr Met Glu Gln Thr Gln Ala Phe Glu Asn Arg Val 20 25 30 Leu Glu Arg Leu Asn Ala Gly Lys Thr Val Arg Ser Phe Leu Ile Thr 35 40 45 Ala Val Glu Leu Leu Thr Glu Ala Val Asn Leu Leu Val Leu Gln Val 50 55 60 Phe Arg Lys Asp Asp Tyr Ala Val Lys Tyr Ala Val Glu Pro Leu Leu 65 70 75 80 Asp Gly Asp Gly Pro Leu Gly Asp Leu Ser Val Arg Leu Lys Leu Ile 85 90 95 Tyr Gly Leu Gly Val Ile Asn Arg Gln Glu Tyr Glu Asp Ala Glu Leu 100 105 110 Leu Met Ala Leu Arg Glu Glu Leu Asn His Asp Gly Asn Glu Tyr Ala 115 120 125 Phe Thr Asp Asp Glu Ile Leu Gly Pro Phe Gly Glu Leu His Cys Val 130 135 140 Ala Ala Leu Pro Pro Pro Pro Gln Phe Glu Pro Ala Asp Ser Ser Leu 145 150 155 160 Tyr Ala Met Gln Ile Gln Arg Tyr Gln Gln Ala Val Arg Ser Thr Met 165 170 175 Val Leu Ser Leu Thr Glu Leu Ile Ser Lys Ile Ser Leu Lys Lys Ala 180 185 190 Phe Gln Lys 195 44 16 PRT Artificial Peptide Sequence 44 Ser Lys His Asp Thr Ser Thr Asn Ala Asn Asp Pro Asn Glu Ser Glu 1 5 10 15 45 14 PRT Artificial Peptide Sequence 45 Gln Asn Lys Ile Arg Gln Asp Gln Ile Asn Asp Ser Asp Thr 1 5 10 46 16 PRT artificial Peptide Sequence 46 Arg Ile Asn Ser Asp Ser Tyr Trp Asp Asn Leu Pro Glu Glu Gln Arg 1 5 10 15 47 14 PRT Artificial Peptide Sequence 47 Thr Leu Val Glu Arg Asp Glu Asn Gly Asn Ser Asn Tyr Gly 1 5 10 48 21 DNA Artificial Primer Sequence 48 ttcataagat gtcacgccag g 21 49 20 DNA artificial Primer Sequence 49 ggtacgcgtc atattccttg 20

Claims

What is claimed:

1. A Brassica sp. plant comprising a heterologous nucleic acid molecule encoding a multifunctional fatty acid synthase.

2. The plant of claim 1, further comprising a second heterologous nucleic acid molecule encoding a phosphopantetheine:protein transferase enzyme.

3. The plant of claim 1 or 2, wherein the nucleic acid molecule encodes a multifunctional fatty acid synthase comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 15, 17, 18, 19, 20, 22-25, 27, 29, and 31.

4. The plant of claim 3, wherein the protein is SEQ ID NO: 2.

5. The plant of claim 2, wherein the nucleic acid molecule encoding a phosphopantetheine:protein transferase enzyme comprises SEQ ID NOs: 4, 33, 35, 37, 39, 41, and 43.

6. The plant of claim 1 or 2, wherein the multifunctional fatty acid synthase increases oil levels in the seed of a plant that has been transformed with said nucleic acid.

7. The plant of claim 2, that further comprises a promoter that is operably linked to the nucleic acid encoding a multifunctional fatty acid synthase or the second nucleic acid encoding a phosphopantetheine:protein transferase enzyme, wherein the promoter is functional in a plant cell.

8. The plant of claim 7, wherein the promoter provides expression substantially within plant seeds of the multifunctional fatty acid synthase or the phosphopantetheine:protein transferase enzyme.

9. The plant of claim 1 or 2, wherein the multifunctional fatty acid synthase is substantially located in the cytosol of a plant cell.

10. A method for producing a Brassica sp. plant with increased oil content that comprises,

a. introducing a first nucleic acid molecule encoding a multifunctional fatty acid synthase into a Brassica sp. plant cell; and

b. regenerating a plant from the plant cell.

11. The method of claim 10, further comprising introducing a second nucleic acid molecule encoding a phosphopantetheine:protein transferase enzyme into the plant cell.

12. The method of claim 10 or 11, wherein the first nucleic acid molecule is isolated from Brevibacterium ammoniagenes (ATCC 5871), Schizosaccharomyces pombe, Saccharomyces cerevisiae, Candida albicans, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium bovis, Caenorhabditis elegans, Lipomyces starkeyi, rat, or chicken.

13. The method of claim 10 or 11, wherein the first nucleic acid molecule encodes a multifunctional fatty acid synthase having an amino acid sequence comprising a protein selected from the group consisting of SEQ ID NOs: 2, 15, 17, 18, 19, 20, 22-25, 27, 29, and 31.

14. The method of claim 13, wherein the protein is SEQ ID NO: 2.

15. The method of claim 11, wherein the second nucleic acid molecule encoding a phosphopantetheine:protein transferase enzyme encodes an amino acid comprising a protein selected from the group consisting of SEQ ID NOs: 4, 33, 35, 37, 39, 41, and 43.

16. The method of claim 10 or 11, wherein the multifunctional fatty acid synthase produces increased oil levels in a seed of the plant.

17. The method of claim 11, wherein the first nucleic acid molecule encoding a multifunctional fatty acid synthase or the second nucleic acid molecule encoding a phosphopantetheine:protein transferase enzyme further comprises a promoter that is operably linked thereto, wherein the promoter is functional in the plant cell.

18. The method of claim 17, wherein the promoter is a globulin promoter, a zein promoter, an oleosin promoter, an ubiquitin promoter, a CaMV ³⁵S promoter, a CaMV 19S promoter, a nos promoter, an Adh promoter, a sucrose synthase promoter, a tubulin promoter, a napin promoter, an actin promoter, a cab promoter, a PEPCase promoter, a 7S-alpha′-conglycinin promoter, an R gene complex promoter, a tomato E8 promoter, a patatin promoter, a mannopine synthase promoter, a soybean seed protein glycinin promoter, a soybean vegetative storage protein promoter, or a root-cell promoter.

19. The method of claim 17, wherein the promoter provides expression substantially within plant seeds of the multifunctional fatty acid synthase or the phosphopantetheine:protein transferase enzyme.

20. The method of claim 10 or 11, wherein the multifunctional fatty acid synthase is substantially located in the cytosol of the plant cell.

21. A method of producing a Brassica oil, comprising the steps of:

a) growing an oilseed Brassica plant, the genome of which contains a nucleic acid molecule encoding a multifunctional fatty acid synthase, to produce oil-containing seeds; and

b) extracting oil from the seeds.

22. A method of producing a Brassica oil, comprising the steps of:

a) growing an oilseed Brassica plant, the genome of which contains a nucleic acid molecule encoding a phosphopantetheine:protein transferase, to produce oil-containing seeds; and

b) extracting oil from the seeds.